CA2390141C

CA2390141C - Novel compositions and processes for improved analyte detection using hybridization assays

Info

Publication number: CA2390141C
Application number: CA2390141A
Authority: CA
Inventors: Elazar Rabbani; Jannis G. Stavrianopoulos; James J. Donegan; Jack Coleman
Original assignee: Enzo Life Sciences Inc
Current assignee: Enzo Life Sciences Inc
Priority date: 2001-06-30
Filing date: 2002-06-10
Publication date: 2011-01-04
Anticipated expiration: 2022-06-10
Also published as: IL201151A; CA2841397C; US20060035238A1; IL210625A; US20160186246A1; US9279147B2; US9650666B2; US9234235B2; IL210637A; IL210630A0; US20160032372A1; US9771667B2; IL210631A0; IL210625A0; CA2390141A1; US20060014156A1; US20150376688A1; EP2361992A1; IL210626A; US9234234B2

Abstract

This invention provides novel compositions and processes for analyte detection, quantification and amplification. Nucleic acid arrays and libraries of analytes are usefully incorporated into such compositions and processes. Universal detection elements, signaling entities and the like are employed to detect and if necessary or desirable, to quantify analytes. Amplification of target analytes are also provided by the compositions and processes of this invention.

Description

Novel Compositions and Processes for Analyte Detection, Quantification and Amplification FIELD OF THE INVENTION
This invention relates to the field of analyte detection, quantification and amplification, including compositions and processes directed thereto.
All patents, patent applications, patent publications, scientific articles and the like, cited or identified in this application are hereby incorporated by reference in their entirety in order to describe more fully the state of the art to which the present invention pertains.
BACKGROUND OF THE INVENTION
The quantification of RNA expression provides major insights into analysis of cellular metabolism, function, growth and interactions. Although individual RNA
species have historically been the subject of these studies, more interest is currently being shown in analysis of the patterns of the simultaneous expression of multiple RNA species of both known and unknown function. This approach allows comparative studies on the patterns of expression between different populations of cells, thereby serving as an indicator of the differences in biochemical activities taking place within these populations. For instance, a single group of cells can be divided up into two or more populations where one group serves as a control and the other part is exposed to drugs, metabolites or different physical conditions. In this way, although the majority of the various species of mRNA show little or no differences in expression levels, certain mRNA species may show dramatic increased or decreased levels of expression compared to the untreated or normal control.
As an example, it has long been known that the application of a phorbol ester (PMA) results in changes in a large number of characteristics of mammalian cells growing in vitro. In an experiment reported by Lockhart et al., ( 1996, Nature Biotechnology 14; 1675-1680) cells growing in culture v~iere exposed to PMA
and at various times afterwards, mRNA was extracted and used to create a library of labeled probes. This material was subsequently hybridized to an array of nucleic Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 2 (New Patent Application) acids that was complementary to various mRNA sequences. Significant changes could be seen in both the timing and the amount of induction of various cellular cytokines. On the other hand, so called "house-keeping" genes such as actin and GAPDH remained essentially unaffected by the treatment. This example demonstrates that the various mRNA's can be independently monitored to determine which particular genes may be affected by a treatment.
Natural differences between cell populations can also be examined. For instance, differences in the expression levels of various genes can be observed when cells progress through cell cycles (Cho et al., 1998 Mol Cell 2; 65-73 and Spellman et al., 1998 Mol. Biol. Cell 95; 14863-14868). The gene expression profiles that were generated by these studies validated this approach when significant differences in expression were observed for genes that had previously been characterized as encoding cell cycle related proteins. In addition, the arrays used in these studies comprised nucleic acid sequences that represented the entire genetic complement of the yeast being studied. As such, one of the results of these studies was the observation of a number of genes of previously unknown function that also displayed cell cycle dependent expression. Re-examination of these particular genes by other more conventional methods demonstrated that they were involved in cell cycle progression. Thus, this method was demonstrated as being capable of recognizing genes previously known for differential expression and also for identifying new genes.
The differences between normal and transformed cells have also been a subject of long standing interest. The nature of the particular genes that are either overexpressed or underexpressed relative to normal cells may provide information on the origination, progression or treatment of cancerous cells. Array analysis has been carried out by using RNA from tumor derived cells iri comparison with expression from normal cells. In one study by Perou et al (1999 Proc. Nat Acad.
Sci. USA 96; 9212-9217) human mammary epithelial cells (HMEC) were compared Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 3 (New Patent Application) with specimens from primary breast tumors. Included in this study were responses to various cell factors as well as the results of confluence or senescence in the control cultures. All of these are factors that may be involved or affected by cellular transformation into the cancerous state. The amount of data generated in this type of study is almost overwhelming in its complexity. However distinct patterns or clusters of expression can be observed that are correlated to factors associated with the specimens. Further understanding will also be gained when data is gathered from expression in other tumor types and their untransformed equivalents.
There are two distinct elements in all of the expression studies that employ arrays. The first element is concerned with the preparation of the bank of probes that will be used to bind or capture labeled material that is derived from the mRNAs that are being analyzed. The purpose of these arrays is to provide a multiplicity of individual probes where each probe is located in a discrete spatially defined position. After hybridization of the sample is carried out, the particular amount of sample is measured for each site giving a relative measurement of how much material is present in the sample that has homology with the particular probe that is located at that site. The two most commonly used methods for array assembly operate on two very different scales for synthesis of arrays.
On the simplest level of construction, discrete nucleic acids are affixed to solid matrixes such as glass slides or nylon membranes in a process that is very similar to that employed by ink jet printers (For example, see Okamoto et al., 2000, Nature Biotechnology 18; 438-441 ). The nature of the probe deposited on the matrix can range from small synthetic oligonucleotides to large nucleic acid segments from clones. Preparation of a cloned segment to be used in this form of array assembly can range from E. coli colonies containing individual clones that are lysed and fixed directly onto a matrix or more elaborately by using individual plasmids as templates for preparation of PCR amplified material. The latter method Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 4 (New Patent Application) is preferred due to the higher purity of the nucleic acid product. The choice of a particular probe to be used in the assembly can be directed in the sense that the function and sequence is known. This of course will always be true when oligonucleotides are used as the probes since they must be synthesized artificially.
On the other hand, when the probes are derived from larger cloned segments of DNA, they can be used irrespective of knowledge of sequence or function. For instance, a bank of probes that represent the entire yeast genome was used in the studies cited earlier on differential expression during cell cycle progression. For human sequences, the burgeoning growth of the human sequencing project has provided a wealth of sequence information that is constantly expanding.
Therefore, a popular source of probes that can be used to detect human transcripts has been Expressed Sequence Tags (ESTs) (Adams et al., 1991 Science 252;
1651-1656). The use of sequences of unknown function has the advantage of a lack of any a priori assumption concerning responsiveness in a comparative study and in fact, the study in itself may serve to identify functionality. At present, filter and glass arrays are commercially available from a number of sources for the analysis of expression from various human tissues, developmental stages and disease conditions. On the other hand, directions for making custom arrays are widely disseminated throughout the literature and over the Internet.
At the other end of the scale in complexity is a process where in situ synthesis of oligonucleotides is carried out directly on a solid matrix using a "masking" technology that is similar to that employed in etching of microcircuits (Pirrung et al., U.S. Patent No. 5,143,854, hereby incorporated by reference).
Since this process can be carried out on a very small microscale, a very large number of different probes can be loaded onto a single "biochip" as a high density array. However, since this method depends upon site-specific synthesis, only oligonucleotides are used and the probes are necessarily of limited size.
Also, since directed sequence synthesis is used, sequence information has to be available for Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 5 (New Patent Application) each probe. An advantage of this system is that instead of a single probe for a particular gene product, a number of probes from different segments can be synthesized and incorporated into the design of the array. This provides a redundancy of information, establishing that changes in levels of a particular transcript are due to fluctuations in the intended target rather than by transcripts with one or more similar sequences. These "biochips" are commercially available as well as the hardware and software required to read them.
Although solid supports such as plastic and glass have been commonly used for fixation of nucleic acids, porous materials have also been used. For example, oligonucleotides were joined to aldehyde groups in polyacrylamide IYershov et al., (1996) Proc Nat. Acad. Sci USA 93; 4913-4918) and agarose (Afanassiev et al.
(2000) Nucl. Acids Res. 28; e66) to synthesize arrays that were used in hybridization assays.
The second element involved in array analysis is the means by which the presence and amount of labeled nucleic acids bound to the various probes of the array will be detected. There are three levels of use of the target mRNA that can provide signal generation. In the first approach, the native RNA itself can be labeled. This has been carried out enzymatically by phosphorylation of fragmented RNA followed by T4 RNA ligase mediated addition of a biotinylated oligomer to the 5' ends (Lockhart et al, 1996). This method has the limitation that it entails an overnight incubation to insure adequate joining of labels to the RNA. For chemical labeling of RNA, the fragments can be labeled with psoralen that has been linked to biotin (Lockhart et al, 19961. This method has the disadvantage that the crosslinking that joins the label to the RNA can also lead to intrastrand crosslinking of target molecules reducing the amount of hybridizable material.
In the second approach, rather than labeling the transcript itself, the RNA is used as a template to synthesize cDNA copies by the use of either random primers or by oligo dT primers. Extension of the primers by reverse transcriptase can be Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 6 (New Patent Application) carried out in the presence of modified nucleotides, thereby labeling all of the nascent cDNA copies. The modified nucleotides can have moieties attached that generate signals in themselves or they may have moieties suitable for attachment of other moieties capable of generation of signals. Examples of groups that have been used for direct signal generation have been radioactive compounds and fluorescent compounds such as fluorescein, Texas red, Cy3 and Cy 5. Direct signal generation has the advantage of simplicity but has the limitation that in many cases there is reduced efficiency for incorporation of the labeled nucleotides by a polymerase. Examples of groups that have been used for indirect signal generation in arrays are dinitrophenol (DNP) or biotin ligands. Their presence is detected later by the use of labeled molecules that have affinities for these ligands. Avidin or strepavidin specifically bind to biotin moieties and antibodies can be used that are specific for DNP or biotin. These proteins can be labeled themselves or serve as targets for secondary bindings with labeled compounds. Alternatively, when the labeled nucleotides contain chemically active substituents such as allylamine modifications, post-synthetic modification can be carried out by a chemical addition of a suitably labeled ester.
The synthesis of a cDNA copy from an mRNA template essentially results in a one to one molar ratio of labeled product compared to starting material. In some cases there may be limiting amounts of the mRNA being analyzed and for these cases, some amplification of the nucleic acid sequences in the sample may be desirable. This has led to the use of the third approach, where the cDNA copy derived from the original mRNA template is in itself used as a template for further synthesis. A system termed "Transcription Amplification System" (TAS) was described (Kwoh, D.Y. and Gingeras, T.R., 1989, Proc. Nat. Acad. Sci., 86, 1177) in which a target specific oligonucleotide is used to generate a cDNA
copy and a second target specific oliganucleotide is used to convert the single stranded DNA into double-stranded form. By inclusion of a T7 promoter sequence into the Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 7 (New Patent Application) first oligonucleotide, the double-stranded molecule can be used to make multiple transcription products that are complementary to the original mRNA of interest.
The purpose of this system was for amplification of a discrete sequence from a pool of various RNA species. No suggestion or appreciation of such a system for the use of non-discrete primer sequences for general amplification was described in this work.
Multiple RNA transcript copies homologous to the original RNA population has been disclosed by van fielder et al. in U.S. Patent No. 5,891,636 where specific reference is given to the utility of such a system for creating a library of various gene products in addition to discrete sequences. Since each individual mRNA molecule has the potential for ultimately being the source of a large number of complementary transcripts, this system enjoys the advantages of linear amplification such that smaller amounts of starting material are necessary compared to direct labeling of the original mRNA or its cDNA copy.
However, the work described in U.S. Patent No. 5,891,636 specifically teaches away from addition of exogenous primers for synthesis of a 2"d strand.
Instead, it discloses the use of oligonucleotide primers for production of only the first strand of cDNA. For synthesis of the second strand, two possible methods were disclosed. In the first method, the nicking activity of RNase H on the original mRNA template was used to create primers that could use the cDNA as a template.
In the second method, DNA polymerase was added to form hairpins at the end of the first cDNA strand that could provide self-priming. The first method has a limitation that RNase H has to be added after the completion of the cDNA
synthesis reaction and a balance of RNase H activity has to be determined to provide sufficient nicking without total degradation of potential RNA primers. The second method requires an extra step of incubation a different polymerase besides the j Reverse Transcriptase and also S 1 nuclease has to be added to eliminate the.
loop in the hairpin structure. In addition, the formation and extension_k~~fordback is a Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 8 (New Patent Application) poorly understood system that does not operate at high efficiency where sequences and amounts of cDNA copies may act as random factors.
In addition to the amplification provided by the use of RNA transcription, PCR has been included in some protocols to carry out synthesis of a library through the use of common primer binding sites at each end of individual sequences (Endege et al., 1999 Biotechniques 26; 542-550, Ying et al., 1999 Biotechniques 27; 410-414). These methods share the necessity for a machine dedicated to thermal cycling.
In addition to binding analytes from a library, the nucleic acids on an array can use the analytes as templates for primer extension reactions. For instance, determination of Single Nucleotide Polymorphisms, (SNP's) has been carried out by the use of a set of primers at different sites on the array that exhibit sequence variations from each other (Pastinen et al., 2000, Genome Research 10; 1031-1042). The ability or inability of a template to be used for primer extension by each set of primers is an indication of the particular sequence variations within the analytes. More complex series of reactions have also been carried out by the use of arrays as platforms for localized amplification as described in U.S. Patent No.
5,641,658 and Weslin et al., 2000, Nature Biotechnology 18; 199-204. In these particular applications of array technology, PCR and SDA were carried out by providing a pair of unique primers for each individual nucleic acid target at each locus of the array. The presence or absence of amplification at each locus of the array served as an indicator of the presence or absence of the corresponding target sequences in the analyte samples.
Despite the accelerated development of the synthesis and use of DNA
microarrays in recent years, the progress in the development of arrays of proteins or other ligands has been significantly slower even though such arrays are an ideal format with which to study gene expression, as well as antibody-antigen, receptor-ligand, protein-protein interactions and other applications. In previous art, protein Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 9 (New Patent Application) arrays have been used for gene expression antibody screening, and enzymatic assays (Lueking et al. ( 1999) Anal. Biochem. 270; 103-1 1 1; de Wildt et al., (2000) Nature Biotechnology 18; 989-994, Arenkov et al., (2000) Analytical Biochemistry 278; 123-131 ). Protein arrays have also been used for high throughput ELISA
assays (Mendoza et al., (1999) Biotechniques 27; 778-788) and for the detection of individual proteins in complex solutions IHaab, et al.; (2001 ) Genome Biology 2;
1-13). However, the use thus far has been limited because of the inherent problems associated with proteins. DNA is extremely robust and can be immobilized on a solid matrix, dried and rehydrated without any loss of activity or function. Proteins, however, are far more difficult to utilize in array formats. One of the main problems of using proteins in an array format is the difficulty of applying the protein to a solid matrix in a form that would allow the protein to be accessible and reactive without denaturing or otherwise altering the peptide or protein. Also, many proteins cannot be dehydrated and must be kept in solution at all times, creating further difficulties for use in arrays.
Some methods which have been used to prepare protein arrays include placing the proteins on a polyacrylamide gel matrix on a glass slide that has been activated by treatment with glutaraldehyde or other reagents (Arenkov, op.
cit.).
Another method has been the addition of proteins to aldehyde coated glass slides, followed by blocking of the remaining aldehyde sites with BSA after the attachment of the desired protein. This method, however, could not be used for small proteins because the BSA obscured the protein. Peptides and small proteins have been placed on slides by coating the slides with BSA and then activating the BSA with N,N'-disuccinimidyl carbonate (Taton et al., (20001 Science 2789, 1763). The peptides were then printed onto the slides and the remaining activated sites were blocked with glycine, Protein arrays have also been prepared on poly-L-Lysine coated glass slides (Haab et al., op. cit.) and agarose coated glass slides (Afanassiev et al., (2000) Nucleic Acids Research 28, e66). "Protein Chips"
are Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 10 (New Patent Application) also commercially available from Ciphergen (Fremont, CA) for a process where proteins are captured onto solid surfaces and analyzed by mass spectroscopy.
The use of oligonucleotides as 'hooks' or 'tags' as identifiers for non-nucleic acid molecules has been described in the literature. For instance, a library of peptides has been made where each peptide is attached to a discrete nucleic acid portion and members of the library are tested for their ability to bind to a particular analyte. After isolation of the peptides that have binding affinities, identification was carried out by PCR to "decode" the peptide sequence (Brenner. and Lerner, ( 1992) Proc. Nat. Acad. Sci. USA 89; 5381-5383, Needels et al., ( 1993) Proc.
Nat. Acad. Sci. USA 90; 10,700-10,704). Nuceleic acid sequences have also been used as tags in arrays where selected oligonucleotide sequences were added to primers used for single nucleotide polymorphism genotyping (Hirschhorn, et al., (2000) Proc. Natl. Acad. Sc. USA, 97; 12164-12169). However, in this case the 'tag' is actually part of the primer design and it is used specifically for SNP
detection using a single base extension assay. A patent application filed by Lohse, et al., (WO 00/32823) has disclosed the use of DNA-protein fusions for protein arrays. In this method, the protein is synthesized from RNA transcripts which are then reverse transcribed to give the DNA sequences attached to the corresponding protein. This system lacks flexibility since the technology specifically relates only to chimeric molecules that comprise a nucleic acid and a peptide or protein.
In addition, the protein is directly derived from the RNA sequence so that the resultant DNA sequence is also dictated by the protein sequence. Lastly, every protein that is to be used in an array requires the use of an in vitro translation system made from cell extracts, a costly and inefficient system for large scale synthesis of multiple probes. The use of electrochemically addressed chips for use with chimeric compositions has also been described by Bazin and Livache 1999 in "Innovation and Perspectives in solid Phase Synthesis & Recombinatorial Libraries"
R. Epton (Ed.) Mayflower Scientific Limited, Birmingham, UK.
Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 11 (New Patent Application) SUMMARY OF THE INVENTION
This invention provides a composition of matter that comprises a library of analytes, the analytes being hybridized to an array of nucleic acids, the nucleic acids being fixed or immobilized to a solid support, wherein the analytes comprise an inherent universal detection target (UDT), and a universal detection element (UDE) attached to the UDT, wherein the UDE generates a signal indicating the presence or quantity of the analytes, or the attachment of UDE to UDT.
This invention also provides a composition of matter that comprises a library of analytes, such analytes being hybridized to an array of nucleic acids, and such nucleic acids being fixed or immobilized to a solid support, wherein the analytes comprise a non-inherent universal detection target (UDT) and a universal detection element (UDE) hybridized to the UDT, and wherein the UDE generates a signal directly or indirectly to detect the presence or quantity of such analytes.
The present invention further provides a composition of matter that comprises a library of analytes, such analytes being hybridized to an array of nucleic acids, and such nucleic acids being fixed or immobilized to a solid support, wherein the hybridization between the analytes and the nucleic acids generate a domain for complex formation, and the composition further comprises a signaling entity complexed to the domain.
The present invention yet further provides a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of: a) providing: (i1 an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of the nucleic acids of interest comprise at least one inherent universal detection target (UDT); and (iii) universal detection elements (UDE) which generates a signal directly or indirectly; b) hybridizing the library (ii) with the array of nucleic acids fi) to form hybrids if the nucleic acids of interest are present; c) contacting Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 12 (New Patent Application) the UDEs with the UDTs to form a complex bound to the array; d) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Also provided by this invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing: (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of the nucleic acids of interest comprise at least one inherent universal detection target (UDT); and (iii) universal detection elements (UDE) which generates a signal directly or indirectly;
b) contacting the UDEs with the UDTs in the library of nucleic acid analytes to form one or more complexes; c) hybridizing the library of nucleic acid analytes with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; d) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Also provided herein is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of the nucleic acids of interest comprise at least one non-inherent universal detection target (UDT), wherein the non-inherent UDT is attached to the nucleic acid analytes; and (iii) universal detection elements (UDE) which generate a signal directly or indirectly; b) hybridizing the library (ii) with the array of nucleic acids Ii) to form hybrids if the nucleic acids of interest are present; c) contacting the UDEs with the UDTs to form a complex bound to the array; d) detecting or quantifying the more than one Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 13 (New Patent Application) nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Another aspect provided by this invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of such nucleic acids of interest comprise at least one non-inherent universal detection target (UDT), wherein the non-inherent UDTs are attached to the nucleic acid analytes; and (iii) universal detection elements (UDE) which generate a signal directly or indirectly; b) contacting the UDEs with the UDTs in the library of nucleic acid analytes to form one or more complexes; c) hybridizing the library (ii) with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; d) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Another aspect provided by this invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more universal detection targets (UDT) to a nucleic acid;
(iv) universal detection elements (UDE) which generates a signal directly or indirectly;
b) attaching such UDTs (iii) to the library of nucleic acid analytes (ii); c) hybridizing the library (ii) with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; d) contacting the UDEs with the UDTs to form a complex bound to the array; e) detecting or quantifying the more than one nucleic acid of Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 14 (New Patent Application) interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Still another feature is process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) means for attaching one or more universal detection targets (UDT) to a nucleic acid; (iv) universal detection elements (UDE) which generate a signal directly or indirectly; b) attaching the UDTs (iii) to the library of nucleic acid analytes (ii); c) contacting the UDEs with the UDTs in the library of nucleic acid analytes to form one or more complexes; d) hybridizing the library (ii) with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; e) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
The present invention provides additionally a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) universal detection elements (UDEs) which bind to a domain formed by nucleic acid hybrids for complex formation and generate a signal directly or indirectly; b) hybridizing the library (ii) with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present, wherein any formed hybrids generate a domain for complex formation; c) contacting the UDEs with any hybrids to form a complex bound to the array; d) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 15 (New Patent Application) Also provided herein is a composition of matter comprising a library of first nucleic acid analyte copies, such first nucleic acid copies being hybridized to an array of nucleic acids, those nucleic acids being fixed or immobilized to a solid support, wherein such first nucleic acid copies comprise an inherent universal detection target (UDT) and a universal detection element (UDE) attached to the UDT, wherein the UDE generates a signal directly or indirectly to detect the presence or quantity of any analytes.
Another embodiment of this invention is a composition of matter comprising a library of first nucleic acid analyte copies, such first nucleic acid copies being hybridized to an array of nucleic acids, the nucleic acids being fixed or immobilized to a solid support, wherein such first nucleic acid copies comprise one or more non-inherent universal detection targets (UDTs) and one or more universal detection elements (UDEs) attached to the UDTs, wherein the UDEs generate a signal directly or indirectly to detect the presence or quantity of any analytes, and wherein the UDTs are either: (i) at the 5' ends of the first nucleic acid copies and not adjacent to an oligoT segment or sequence, or (ii) at the 3' ends of the first nucleic acid copies, or (iii) both (i) and (ii).
This invention also concerns a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of such nucleic acids of interest comprise at least one inherent universal detection target (UDT); (iii) universal detection elements (UDE) which generate a signal directly or indirectly; and (iv) polymerizing means for synthesizing nucleic acid copies of the nucleic acids of analytes; b) synthesizing one or more first nucleic acid copies which are complementary to all or part of the nucleic acid analytes and synthesizing sequences which are complementary to all or part of the UDT to form Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 16 (New Patent Application) a complementary UDT; c) hybridizing such first nucleic acid copies with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; d) contacting the UDEs with the complementary UDTs of the first nucleic acid copies to form a complex bound to the array; e) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Another embodiment provided by this invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of such nucleic acids of interest comprise at least one inherent universal detection target (UDT); (iii) universal detection elements (UDE) which generate a signal directly or indirectly; and (iv) polymerizing means for synthesizing nucleic acid copies of such nucleic acid analytes; b) synthesizing one or more first nucleic acid copies of such nucleic acid analytes; c) contacting the UDEs with the UDTs in the first nucleic acid copies to form one or more complexes; d) hybridizing such first nucleic acid copies with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; and e) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
An additional aspect of the present invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid; (iv) universal detection elements (UDE) which generate a signal Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 17 (New Patent Application) directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes; b) attaching the non-inherent UDTs to either the 3' ends of the nucleic acid analytes, the 5' ends of the first nucleic acid analytes, or both the 3' ends and the 5' ends of the nucleic acid analytes; c) synthesizing one or more first nucleic acid copies of the nucleic acid analytes; d) hybridizing the first nucleic acid copies with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; e) contacting the UDEs with the UDTs of the first nucleic acid copies to form a complex bound to the array; f) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Also provided herein is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iiit means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid; (iv) universal detection elements (UDE) which generate a signal directly or indirectly;
and (v) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes; b) attaching such non-inherent UDTs to either the 3' ends of the nucleic acid anaiytes, the 5' ends of the first nucleic acid analytes, or both the 3' ends and the 5' ends of the nucleic acid analytes; c) synthesizing one or more first nucleic acid copies of the nucleic acid analytes; d) contacting the UDEs with the UDTs of the first nucleic acid copies to form complexes; e) hybridizing the first nucleic acid copies with the array of nucleic acids li) to form hybrids if any nucleic acids of interest are present; f) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 1$ (New Patent Application) Another embodiment provided herein is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to such nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid; (iv) universal detection elements (UDE) which generate a signal directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes; b) synthesizing one or more first nucleic acid copies of the nucleic acid analytes; c) attaching the non-inherent UDTs to either the 3' ends of the first nucleic acid copies, the 5' ends of the first nucleic acid copies, or both the 3' ends and the 5' ends of the first nucleic acid copies; d) hybridizing the first nucleic acid copies with the array of nucleic acids Ii) to form hybrids if any nucleic acids of interest are present; e) contacting the UDEs with the UDTs of the first nucleic acid copies to form a complex bound to the array; and f) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Another process provided by this invention is for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid; (iv) universal detection elements (UDE) which generate a signal directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes; b) synthesizing one or more first nucleic acid copies of the nucleic acid analytes; c) attaching the non-inherent UDTs to either the 3' ends of the first nucleic acid copies, the 5' ends of the first nucleic acid copies, Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 19 (New Patent Application) or both the 3' ends and the 5' ends of the first nucleic acid copies; d) contacting the UDEs with the UDTs of the first nucleic acid copies to form a complex; e) hybridizing the first nucleic acid copies with the array of nucleic acids (i) to form hybrids if any nucleic acids of interest are present; and f) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Yet further provided is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) universal detection elements (UDEs) which bind to a domain for complex formation formed by nucleic acid hybrids and generate a signal directly or indirectly; and (iv) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes; b) synthesizing one or more nucleic acid copies of the nucleic acid analytes; c) hybridizing the first nucleic acid copies with the array of nucleic acids (i) to form hybrids if any nucleic acids of interest are present, wherein any formed hybrids generate a domain for complex formation; d) contacting the UDEs with the hybrids to form a complex bound to the array; and e) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Another aspect provided by this invention is a composition of matter comprising a library of double-stranded nucleic acids substantially incapable of in vivo replication and free of non-inherent homopolymeric sequences, the nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample, wherein the double-stranded nucleic acids comprise at least one inherent universal detection target (UDT) proximate to Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 20 (New Patent Application) one end of the double strand and at least one non-inherent production center proximate to the other end of the double strand.
Yet another aspect of this invention concerns a composition of matter comprising a library of double-stranded nucleic acids substantially incapable of in vivo replication, such nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample, wherein the double-stranded nucleic acids comprise at least four (4) non-inherent nucleotides proximate to one end of the double strand and a non-inherent production center proximate to the other end of the double strand.
Among other useful aspects of this invention is a composition of matter comprising a library of double-stranded nucleic acids fixed to a solid support, those nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample and the nucleic acids further comprising at feast one first sequence segment of non-inherent nucleotides proximate to one end of the double strand and at least one second sequence segment proximate to the other and of the double strand, the second sequence segment comprising at least one production center.
Another feature of this invention is a composition of matter comprising a library of double-stranded nucleic acids attached to a solid support, the nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample, wherein the double-stranded nucleic acids comprise at least one inherent universal detection target (UDT) proximate to one end of the double strand and at least one non-inherent production center proximate to the other end of the double strand.
The invention herein also provides a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest;
(ii) a Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 21 (New Patent Application) library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, the polymerizing means comprising a first set of primers and a second set of primers, wherein the second set of primers comprises at least two segments, the first segment at the 3' end comprising random sequences, and the second segment comprising at least one production center; (iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions; b) contacting the library of nucleic acid anafytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) contacting the extended first copies with the second set of primers to form more than one second bound entity;
e1 extending the bound second set of primers by means of template sequences provided by the extended first copies to farm more than one complex comprising extended first copies and extended second set of primers; f) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; g) hybridizing any nucleic acid copies formed in step f) to the array of nucleic acids provided in step a1 (i); and h) detecting or quantifying any of the hybridized copies obtained in step 9) Also provided by this invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; Iii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of prirners and a second set of primers, wherein the first set of primers comprise at least one Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 22 (New Patent Application) production center; and (iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions; b) contacting the library of nucleic acid analytes with the first set of primers to farm more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) extending the first copies by means of at least four (4) or more non-inherent hornopolymeric nucleotides; e) contacting the extended first copies with the second set of primers to form more than one second bound entity; f) extending the bound second set of primers by means of template sequences provided by the extended first copies to form more than one complex comprising extended first copies and extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of the hybridized copies obtained in step h).
Another feature of this invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing Ii) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the first set comprises at least one production center; (iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of the second set of primers; and(v) means for ligating the set of oligonucleotides or polynucleotides (iv); b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 23 (New Patent Application) first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) ligating the set of oligonucleotides or polynucleotides a) (iv) to the 3' end of the first copies formed in step c) to form more than one ligated product; e) contacting the ligated product with the second set of primers to form more than one second bound entity; f) extending the bound second set of primers by means of template sequences provided by the ligated products formed in step d) to form more than one complex comprising the ligated products and the extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of the hybridized copies obtained in step h).
Still yet further this invention provides a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i1 an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the second set comprises at least one production center; (iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of the second set of primers; and (v) means for ligating the set of oligonucleotides or polynucleotides (iv); b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) ligating the set of oligonucleotides Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 24 (IVew Patent Application) or polynucleotides a) (iv) to the 3' end of the first copies formed in step c) to form more than one ligated product; e) contacting the ligated product with the second set of primers to form more than one second bound entity; f) extending the bound second set of primers by means of template sequences provided by the ligated products formed in step d) to form more than one complex comprising the ligated products and the extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step a) ti); and i) detecting or quantifying any of the hybridized copies obtained in step h).
Still yet further provided by this invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers, a second set of primers and a third set of primers wherein the third set comprises at least one production center; and b) contacting the library of nucleic acid analytes with the first set of primers to form a first set of bound primers; c) extending the first set of bound primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) contacting the extended first copies with the second set of primers to form a second set of bound primers; e) extending the second set of bound primers by means of template sequences provided by the extended first copies to form second copies of the nucleic acid analytes; f) contacting the second copies with the third set of primers to form more than one third bound entity to form a third set Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 25 (New Patent Application) of bound primers; g) extending the third set of bound primers by means of template sequences provided by the extended second set of primers to form a hybrid comprising a second copy, a third copy and at least one production center; h) synthesizing from the production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions;
i) hybridizing the nucleic acid copies formed in step i) to the array of nucleic acids provided in step a) (i); and j) detecting or quantifying any of the hybridized copies obtained in step i).
Also uniquely provided in this invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing Ii) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the first set of primers are fixed or immobilized to a solid support, and wherein the second set of primers comprises at least two segments, the first segment at the 3' end comprising random sequences, and the second segment comprising at least one production center; (iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions; b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) contacting the extended first copies with the second set of primers to form more than one second bound entity;
e) extending the bound second set of primers by means of template sequences provided by the extended first copies to form more than one complex comprising extended first copies and extended second set of primers; f) synthesizing from a Enz-60 Elazar Rabbani et al., f-mng Date: Herewith Page 26 (New Patent Application) production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; g) hybridizing the nucleic acid copies formed in step f) to the array of nucleic acids provided in step a) (i); and h) detecting or quantifying any of the hybridized copies obtained in step 9).
Another significant aspect of this invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the first set of primers are fixed or immobilized to a solid support, and wherein the first set of primers comprise at least one production center; and (iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions; b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) extending the first copies by means of at least four (4) or more non-inherent homopolymeric nucleotides; e) contacting the extended first copies with the second set of primers to form more than one second bound entity; f) extending the bound second set of primers by means of template sequences provided by the extended first copies to form more than one complex comprising extended first copies and extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; h) hybridizing the nucleic acid Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 27 (New Patent Application) copies formed in step g) to the array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of the hybridized copies obtained in step h).
Also provided in accordance with the present invention is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the first set of primers are fixed or immobilized to a solid support, and wherein the first set comprises at least one production center; (iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of the second set of primers; and (v) means for ligating the set of oligonucleotides or polynucleotides (iv); b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first. set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d1 ligating the set of oligonucleotides or polynucleotides a) (iv) to the 3' end of the first copies formed in step c) to form more than one ligated product; e) contacting the ligated product with the second set of primers to form more than one second bound entity; f) extending the bound second set of primers by means of template sequences provided by the ligated products formed in step d) to form more than one complex comprising the ligated products and the extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step Enz-60 Elazar Rabbani et al., Fning Date: Herewith Page 28 (New Patent Application) a) (i); and i) detecting or quantifying any of the hybridized copies obtained in step hl.
Another feature of the present invention concerns a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest;
Iii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the first set of primers are fixed or immobilized to a solid support, and wherein the second set comprises at least one production center; (iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of the second set of primers; and (v) means for ligating the set of oligonucleotides or polynucleotides (iv); b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) ligating the set of oligonucleotides or polynucleotides a) (iy) to the ~' end of the first copies formed in step c) to form more than one ligated product; e) contacting the ligated product with the second set of primers to form more than one second bound entity; f) extending the bound second set of primers by means of template sequences provided by the ligated products formed in step d) to form more than one complex comprising the ligated products and the extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of the hybridized copies obtained in step h).
Enz-60 Elazar Rabbani et al., Fmng Date: Herewith Page 29 (New Patent Application) Yet another process is provided by this invention, the process being one for detecting or quantifying more than one nucleic acid of interest in a library and comprising the steps of a) providing (l) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers, a second set of primers and a third set of primers, wherein the first set of primers are fixed or immobilized to a solid support, and wherein the third set comprises at least one production center; and b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes .to form first copies of the analytes; d) contacting the extended first copies with the second set of primers to form more than one second bound entity; e) extending the bound second set of primers by means of template sequences provided by the extended first copies to form an extended second set of primers; f) separating the extended second set of primers obtained in step e); g) contacting the extended second set of primers with the third set of primers to form more than one third bound entity; h) extending the third bound entity by means of template sequences provided by the extended second set of primers to form more than one complex comprising the extended third bound entity and the extended set of primers; l) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; j) hybridizing the nucleic acid copies formed in step l) to the array of nucleic acids provided in step a) (l); and k) detecting or quantifying any of the hybridized copies obtained in step j).
Another significant embodiment provided herein is a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 30 (New Patent Application) of a) providing 1i) an array of fixed or immobilized nucleic acids identical in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers; b) contacting the nucleic acid analytes with the first set of primers to form a first bound entity; c) extending the bound set of first set of primers by means of template sequences provided by the nucleic acid analytes to form first nucleic acid copies of the analytes; d) separating the first nucleic acid copies from the analytes;
e) repeating steps b), c) and d) until a desirable amount of first nucleic acid copies have been synthesized; f) hybridizing the nucleic nucleic acid copies formed in step e) to the array of nucleic acids provided in step (i1; and g) detecting or quantifying any of the hybridized first nucleic acid copies obtained in step fl.
The invention described herein also provides a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers; (iv) means for addition of sequences to the 3' end of nucleic acids; b) contacting the nucleic acid analytes with the first set of primer to form a first bound entity; c) extending the bound set of first set of primers by means of template sequences provided by the nucleic acid analytes to form first nucleic acid copies of the analytes; d) extending the first nucleic copies by the addition of non-template derived sequences to the 3' end of the first nucleic acid copies; e) contacting the extended first nucleic acid copies with the second set of primers to form a second bound entity; f) extending the bound set of second set of Enz-60 Elazar Rabbani et al., h~nng Dates Herewith Page 31 (New Patent Application) primers by means of template sequences provided by the extended first nucleic acid copies to form second nucleic acid copies; g) separating the second nucleic acid copies from the extended first nucleic acid copies; h) repeating steps e), f) and g) until a desirable amount of second nucleic acid copies have been synthesized; i) hybridizing the second nucleic acid copies formed in step h) to the array of nucleic acids provided in step (i); and j) detecting or quantifying any of the hybridized second nucleic acid copies obtained in step i).
Among other significant compositions provided by the present invention is a composition of matter that comprises an array of solid surfaces comprising discrete areas, wherein at least two of the discrete areas each comprises a first set of nucleic acid primers; and a second set of nucleic acid primers; wherein the nucleotide sequences in the first set of nucleic acid primers are different from the nucleotide sequences in the second set of nucleic acid primers; wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical.
A related composition of this invention concerns a composition of matter that comprises an array of solid surfaces comprising a plurality of discrete areas;
wherein at least two of the discrete areas each comprises a first set of nucleic acid primers; and a second set of nucleic acid primers; wherein the nucleotide sequences in the first set of nucleic acid primers are different from the nucleotide sequences in the second set of nucleic acid primers; wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ substantially from each other; and wherein the nucleotide sequences of the Enz-60 Elazar Rabbani et al., Fmng Date: Herewith Page 32 (New Patent Application) second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical.
Related to the last-mentioned compositions are processes for producing two or more copies of nucleic acids of interest in a library comprising the steps of a) providing (i) an array of solid surfaces comprising a plurality of discrete areas;
wherein at least two of the discrete areas each comprises: (1 ) a first set of nucleic acid primers; and (2) a second set of nucleic acid primers; wherein the nucleotide sequences in the first set of nucleic acid primers are different from the nucleotide sequences in the second set of nucleic acid primers; wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acids of interest; b) contacting a primer of the first set with a complementary sequence in the nucleic acid of interest; c) extending the primer in the first set using the nucleic acid of interest as a template to generate an extended first primer; d) contacting a primer in the second set with a complementary sequence in the extended first primer; e) extending the primer in the second set using the extended first primer as a template to generate an extended second primer; f) contacting a primer in the first set with a complementary sequence in the extended second primer; g) extending the primer in the first set using the extended second primer as a template to generate an extended first primer; and h) repeating steps d) through g) above one or more times.
Enz-60 Elazar Rabbani et al., Fnmg Date: Herewith Page 33 (New Patent Application) Another related process of the present invention is useful for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing (i) an array of solid surfaces comprising a plurality of discrete areas;
wherein at least two of such discrete areas each comprises: ( 1 ) a first set of nucleic acid primers; and (2) a second set of nucleic acid primers; wherein the nucleotide sequences in the first set of nucleic acid primers are different from the nucleotide sequences in the second set of nucleic acid primers; wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acids of interest;
and (iv) non-radioactive signal generating means capable of being attached to or incorporated into nucleic acids; b) contacting a primer of the first set with a complementary sequence in the nucleic acid of interest; c) extending the primer in the first set using the nucleic acid of interest as a template to generate an extended first primer; d) contacting a primer in the second set with a complementary sequence in the extended first primer; e) extending the primer in the second set using the extended first primer as a template to generate an extended second primer; f) contacting a primer in the first set with a complementary sequence in the extended second primer; g) extending the primer in the first set using the extended second primer as a template to generate an extended first primer; h) repeating steps d) through g) above one or more times;
and i) detecting or quantifying by means of the non-radioactive signal generating Enz-60 Elazar Rabbani et al., f-~nng Date: Herewith Page 34 (New Patent Application) means attached to or incorporated into any of the extended primers in steps c), e), g), and h).
Another useful composition provided by the present invention is a composition of matter that comprises an array of solid surfaces comprising a plurality of discrete areas, wherein at least two of such discrete areas comprise: a chimeric composition comprising a nucleic acid portion; and a non-nucleic acid portion, wherein the nucleic acid portion of a first discrete area has the same sequence as the nucleic acid portion of a second discrete area, and wherein the non-nucleic acid portion has a binding affinity for analytes of interest.
Further provided by the present invention is a composition of matter that comprises an array of solid surfaces comprising a plurality of discrete areas;
wherein at least two of the discrete areas comprise a chirneric composition hybridized to complementary sequences of nucleic acids fixed or immobilized to the discrete areas, wherein the chimeric composition comprises a nucleic acid portion, and a non-nucleic acid portion, the nucleic acid portion comprising at least one sequence, wherein the non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when the non-nucleic acid portion is a peptide or protein, the nucleic acid portion does not comprises sequences which are either identical or complementary to sequences that code for such peptide or protein.
Also provided as a significant aspect of the present invention is a process for detecting or quantifying analytes of interest, the process comprising the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas, wherein at least two of such discrete areas comprise a chimeric composition comprising a nucleic acid portion, and a non-nucleic acid portion; wherein the nucleic acid portion of a first discrete area has the same sequence as the nucleic acid portion of a second discrete area; and wherein the non-nucleic acid portion has a binding affinity for analytes of interest; b) a sample containing or suspected of containing one or more of the analytes of interest; and c) signal generating means;
Enz-60 Elazar Rabbani et al., Fmng Date: Herewith Page 35 (New Patent Application)

2) contacting the array a) with the sample b) under conditions permissive of binding the analytes to the non-nucleic acid portion; 3) contacting the bound analytes with the signal generating means; and 4) detecting or quantifying the presence of the analytes.
Another feature provided by the present invention is a process for detecting or quantifying analytes of interest, this process comprising the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas;
wherein at least two of such discrete areas comprise a chimeric composition comprising a nucleic acid portion; and a non-nucleic acid portion; wherein the nucleic acid portion of a first discrete area has the same sequence as the nucleic acid portion of a second discrete area; and wherein the non-nucleic acid portion has a binding affinity for analytes of interest; b) a sample containing or suspected of containing one or more of the analytes of interest; and c) signal generating means; 2) labeling the analytes of interest with the signal generating means; 3) contacting the array a) with the labeled analytes under conditions permissive of binding the labeled analytes to the non-nucleic acid portion; and 4) detecting or quantifying the presence of the analytes.
Also provided by the present invention is a process for detecting or quantifying analytes of interest, the process comprising the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas; Wherein at least two of such discrete areas comprise nucleic acids fixed or immobilized to such discrete areas, b) chimeric compositions comprising: i) a nucleic acid portion.; and ii) a non-nucleic acid portion; the nucleic acid portion comprising at least one sequence, wherein the non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when the non-nucleic acid portion is a peptide or protein, the nucleic acid portion does not comprise sequences which are either identical or complementary to sequences that code for the peptide or protein; c) a sample containing or suspected of containing the analytes of interest; and d) signal Enz-60 Elazar Rabbani et al., ~~nng Date: Herewith Page 36 (New Patent Application) generating means; 2) contacting the array with the chimeric compositions to hybridize the nucleic acid portions of the chimeric compositions to complementary nucleic acids fixed or immobilized to the array; 3) contacting the array a) with the sample b) under conditions permissive of binding the analytes to the non-nucleic acid portion; 4) contacting the bound analytes with the signal generating means;
and 5) detecting or quantifying the presence of the analytes.
Additionally this invention provides a process for detecting or quantifying analytes of interest, the process comprising the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of the discrete areas comprise nucleic acids fixed or immobilized to the discrete areas, b) chimeric compositions comprising i) a nucleic acid portion; and ii) a non-nucleic acid portion, the nucleic acid portion comprising at least one sequence, wherein the non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when the non-nucleic acid portion is a peptide or protein, the nucleic acid portion does not comprise sequences which are either identical or complementary to sequences that code for the peptide or protein; c) a sample containing or suspected of containing the analytes of interest; and d) signal generating means; 2) contacting the chimeric compositions with the sample b) under conditions permissive of binding the analytes to the non-nucleic acid portion; 3) contacting the array with the chimeric compositions to hybridize the nucleic acid portions of the chimeric compositions to complementary nucleic acids fixed or immobilized to the array; 4) contacting the bound analytes with the signal generating means; and 5) detecting or quantifying the presence of the analytes.
Another useful provision of the invention herein is a process for detecting or quantifying analytes of interest, such process comprising the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas;
wherein at least two of the discrete areas comprise nucleic acids fixed or immobilized to the discrete areas, b) chimeric compositions comprising i) a nucleic acid portion;
and ii) Enz-60 Elazar Rabbani et al., Fmng Date: Herewith Page 37 (New Patent Application) a non-nucleic acid portion; the nucleic acid portion comprising at least one sequence, wherein the non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when the non-nucleic acid portion is a peptide or protein, the nucleic acid portion does not comprise sequences which are either identical or complementary to sequences that code for the peptide or protein; c) a sample containing or suspected of containing the analytes of interest; and d) signal generating means; 2) contacting the array with the chimeric compositions to hybridize the nucleic acid portions of the chimeric compositions to complementary nucleic acids fixed or immobilized to the array; 3) labeling the analytes of interest with the signal generating means; 4) contacting the array with the labeled analytes to bind the analytes to the non-nucleic acid portion; and 5) detecting or quantifying the presence of the analytes.
Yet further provided by the present invention is a process for detecting or quantifying analytes of interest, the process comprising the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of the discrete areas comprise nucleic acids fixed or immobilized to the discrete areas, b1 chimeric compositions comprising: i) a nucleic acid portion; and ii) a non-nucleic acid portion; the nucleic acid portion comprising at least one sequence, wherein the non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when the non-nucleic acid portion is a peptide or protein, such nucleic acid portion does not comprise sequences which are either identical or complementary to sequences that code for the peptide or protein; c) a sample containing or suspected of containing the analytes of interest; and d) signal generating means; 2) contacting the array with the chimeric compositions to hybridize the nucleic acid portions of the chimeric compositions to complementary nucleic acids fixed or immobilized to the array; 3) labeling the analytes of interest with the signal generating means; 4) contacting the array with the labeled analytes to bind the analytes to the non-nucleic acid portion; and 5) detecting or quantifying Enz-60 Elazar Rabbani et al., I-~~~ng Date: Herewith Page 38 (New Patent Application) the presence of the analytes.
Enz-60 Elazar Rabbani et al., I-~nng Date: Herewith Page 39 (New Patent Application) BRIEF DESCRIPTION OF THE FIGIzlRES
FIGURE 1 shows an array with mRNA from a library of analytes with UDTs.
FIGURE 2 shows fragmentation of analytes followed by addition of non-inherent UDTs to analytes.
FIGURE 3 depicts the incorporation of a non-inherent UDT to a 1 st cNA copy by means of a primer.
FIGURE 4 illustrates the use of Random Primers with Production Centers for 2"d strand synthesis.
FIGURE 5 relates to the same process as FIGURE 4 wherein.the Production Centers are double-stranded.
FIGURE 6 illustrates 2nd cNA strand priming at terminal and internal sites.
FIGURE 7 illustrates 2nd cNA strand priming after Terminal transferase addition of homopolymeric sequences.
FIGURE 8 shows the addition of primer binding sites by ligation.
FIGURE 9 illustrates multiple additions of primer binding sites.
FIGURE 10 shows 1 st strand synthesis by extension of an oligo dT primer bound to a bead followed by 2nd cNA strand synthesis with random primers having production centers.
FIGURE 11 illustrates 1 st strand synthesis from poly T primer indirectly bound to a bead followed by 2nd strand synthesis with random primers having production center.
FIGURE 12 shows the incorporation of a promoter during 3rd strand synthesis.
FIGURE 13 illustrates the synthesis of an amplicon for isothermal amplification of a library of analytes.
FIGURE 14 shows the synthesis of an amplicon for SDA amplification.
Enz-60 Elazar Rabbani et al., Fmng Date: Herewith Page 40 (New Patent Application) FIGURE 15 shows the ligation of a primer binding site for isothermal amplification.
FIGURE 16 shows the binding of an analyte to an array with SPEs and UPEs for solid phase amplification.
FIGURE 17 shows the extension of an SPE on an array during solid phase amplification.
FIGURE 18 shows the binding of an UPE to an extended SPE followed by extension of the UPE during solid phase amplification.
FIGURE 19 shows solid phase amplification in which binding of extended SPEs and UPEs- to unextended SPEs and UPEs occur.
FIGURE 20 depicts an amplification array for comparative analysis.
FIGURE 21 illustrates the use of an array with SPEs and UPEs for SNP
analysis.
FIGURE 22 relates to binding of analytes to SPEs on an array.
FIGURE 23 shows the binding of primers to extended SPEs on an array.
FIGURE 24 demonstrates the binding of primers and extended primers to SPEs on an array.
FIGURE 25 shows the extension of primers and SPEs on an array in accordance with amplification disclosed in this invention.
FIGURE 26 depicts the binding of nucleic acid portions of chirneric compositions to complementary sequences on an array FIGURE 27 is a gel analysis illustrating the dependency on Reverse Transcriptase for the amplification of a library in accordance with this invention and Example 3 below.
FIGURE 28 is a gel analysis that demonstrates transcription after multiple rounds of 2nd strand synthesis as described further below in Example 4.
FIGURE 29 is also a gel analysis that shows second round of RNA
transcription from a library as described in Example 5 below.
Enz-60 Elazar Rabbani et al., Fmng Date: Herewith Page 41 (New Patent Application) FIGURE 30 is a gel analysis also shows transcription from library made after poly dG tailing in accordance with the present invention and Example fi below.
FIGURE 31 is a gel analysis that shows RNA transcription after a series of reactions one of which was 2nd strand synthesis by thermostable DNA
polymerases as described in Example 9 below.
FIGURE 32 is a gel analysis that shows transcription from libraries made from sequential synthesis of 2nd strands as further described in Example 10 below.
FIGURE 33 is also a gel analysis of amplification of a library of analytes using various reverse transcriptases for 1 st stand synthesis Enz-60 Elazar Rabbani et al., F~~ing Date: Herewith Page 42 (New Patent Application) DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses novel methods, compositions and kits that can be used in making and analyzing a library of nucleic acids. The nucleic acids in the sample being tested can be used directly for signal generation or they can be used as templates to provide one or more nucleic acid copies that comprise sequences that are either identical or complementary to the original sequences.
In the present invention the following terms are used and defined below:
An analyte is a biological polymer or ligand that is isolated or derived from biological sources such as organs, tissues or cells, or non-biological sources by _ synthetic or enzymatic means or processes. Examples of biological polymers can include but are not limited to oligonucleotides, polynucleotides, oligopeptides, polypeptides, oligosaccharides, polysaccharides and lipids. Examples of ligands can include but are not necessarily limited to non-peptide antigens, hormones, enzyme substrates, vitamins, drugs, and non-peptide signal molecules.
A library is a diverse collection of nucleic acids that comprises: a) analytes;
b) nucleic acids derived from analytes that comprise sequences that are complementary to sequences in the analytes; c) nucleic acids derived from analytes that comprise sequences that are identical to sequences in the analytes; and d) any combination of the foregoing.
A label is any moiety that is capable of directly or indirectly generating a signal.
A production center is a segment of a nucleic acid or analogue thereof that is capable of producing more than one copy of a sequence that is identical or complementary to sequences that are operably linked to the production center.
Universal Detection Targets (UDTs) are defined as common or conserved segments in diverse nucleic acids that are present in populations of nucleic acids in a sample and are capable of recognition by a corresponding binding partner.
The UDTs may be intrinsic or they may be artificially incorporated into nucleic acids.
Enz-60 Elazar Rabbani et al., Fnrng Date: Herewith Page 43 (New Patent Application) Examples of inherent UDTs can comprise but not be limited to 3' poly A
segments, 6' caps, secondary structures and consensus sequences. Examples of inherent consensus sequences that might find use in the present invention can comprise but not be limited to signal sites for poly A addition, splicing elements and multicopy repeats such as Alu sequences. UDTs may also be artificially incorporated into nucleic acids by an addition to the original analyte nucleic acid or during synthesis of nucleic acids that comprise sequences that are identical ar complementary to the sequences of the original analytes. Artificially added UDTs may be labeled themselves or they may serve as binding partners.
Universal Detection Elements (UDEs) are comprised of two segments: a first segment that is capable of acting as a binding partner for a UDT and a second segment that is either labeled or otherwise capable of generating a detectable Signal. In some cases the first and second segments can be overlapping or even comprise the same segments. When UDEs are labeled, they may comprise a single signal moiety or they may comprise more than one signal entity. Segments of UDEs involved in binding to UDTs or signal generation may comprise but not be limited to polymeric substances such as nucleic acids, nucleic acid analogues, polypeptides, polysacharides or synthetic polymers.
The present invention discloses the use of UDTs and UDEs for the purpose of array analysis. The present invention also discloses novel methods for incorporation of production centers into nucleic acid libraries that may be used in array analysis. These production centers may provide amplification of sequences that are identical or complementary to sequences in the original diverse nucleic acid analytes. The products derived from these production centers may be labeled themselves or UDTs may be incorporated for detection purposes. Nucleic acids that may be of use in the present invention can comprise or be derived from DNA
or RNA. The original population of nucleic acids may comprise but not be limited to genomic DNA, unspliced RNA, mRNA, rRNA and snRNA.
Enz-60 Elazar Rabbani et al., E ",~ng Date: Herewith Page 44 (New Patent Application) This invention provides a composition of matter that comprises a library of analytes, the analytes being hybridized to an array of nucleic acids, the nucleic acids being fixed or immobilized to a solid support, wherein the analytes comprise an inherent universal detection target (UDT), and a universal detection element DUDE) attached to the UDT, wherein the UDE generates a signal indicating the presence or quantity of the analytes, or the attachment of UDE to UDT. The library of analytes can be derived from a biological source selected from the group consisting of organs, tissues and cells, or they may be from non-natural sources as discussed in the definitions section above. Biological analytes can be selected from _ the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing. The nucleic acid array can be selected from the group consisting of DNA, RNA and analogs thereof, an example of the latter being PNA. Such nucleic acids or analogs can be modified on any one of the sugar, phosphate or base moieties. The solid support can take a number of different forms, including being porous or non-porous. A porous solid support can be selected from the group consisting of polyacrylamide and agarose.
A non-porous solid support may comprise glass or plastic. The solid support can also be transparent, translucent, opaque or reflective.
Nucleic acids can be directly or indirectly fixed or immobilized to the solid support. In terms of indirect attachment, the nucleic acids can be indirectly fixed or immobilized to the solid support by means of a chemical linker or linkage arm.
As discussed elsewhere in this disclosure, the inherent UDT can selected from the group consisting of 3' polyA segments, 5' caps, secondary structures, consensus sequences and a combination of any of the foregoing. The consensus sequences can be selected from the group consisting of signal sequences for polyA
addition, splicing elements, multicopy repeats and a combination of any of the foregoing. As also discussed elsewhere in this disclosure, the UDEs can be selected from the group consisting of nucleic acids, nucleic acid analogs, Enz-60 Elazar Rabbani et al., I-~,~ng Date: Herewith Page 45 (New Patent Application) polypeptides, polysaccharides, synthetic polymers and a combination of any of the foregoing. As mentioned previously, such analogs can take the form of PNA. The UDE generates a signal directly or indirectly. Direct signal generation can take any number of forms and can be selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing. Where indirect signal generation is desired, such can take a number of different forms and in this regard can be selected from the group consisting of _ an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing. Among suitable enzymes which can be indirectly detected, these would include enzymes which catalyze any reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.
This invention also provides a composition of matter that comprises a library of analytes, such analytes being hybridized to an array of nucleic acids, and such nucleic acids being fixed or immobilized to a solid support, wherein the analytes comprise a non-inherent universal detection target (UDT) and a universal detection element (UDEI hybridized to the UDT, and wherein the UDE generates a signal directly or indirectly to detect the presence or quantity of such analytes.
The nature of the analyte, the nucleic acid array, modifications, solid support are as described in the preceding paragraphs above. The non-inherent universal detection targets (UDTs) can comprise homopolymeric sequences or heteropolymeric sequences. The universal detection elements (UDEs) can be selected from the group consisting of nucleic acids, nucleic acid analogs and modified forms thereof.
'the UDEs generate a signal directly or indirectly, such direct and indirect signal generation also being discussed in the paragraphs just above.
Enz-60 Elazar Rabbani et al., I-«mg Date: Herewith Page 46 (New Patent Application) The present invention further provides a composition of matter that comprises a library of analytes, such analytes being hybridized to an array of nucleic acids, 'and such nucleic acids being fixed or immobilized to a solid support, wherein the hybridization between the analytes and the nucleic acids generate a domain for complex formation, and the composition further comprises a signaling entity complexed to the domain. Statements and features regarding the nature of the library of analytes, the nucleic acid array, the solid support and fixation or immobilization thereto, and direct/indirect signal generation are as discussed hereinabove, particularly the last several paragraphs. Notably, the domain for complex formation can be selected from the group consisting of DNA-DNA
hybrids, DNA-RNA hybrids, RNA-RNA hybrids, DNA-PNA hybrids and RNA-PNA hybrids.
The signaling entity that is complexed to the domain can be selected from the group consisting of proteins and intercalators. Such proteins .can comprise nucleic acid binding proteins which bind preferentially to double-stranded nucleic acid, the latter comprising antibodies, for example. These antibodies are specific for nucleic acid hybrids and are selected from the group consisting of DNA-DNA hybrids, DNA-RNA hybrids, RNA-RNA hybrids, DNA-PNA hybrids and RNA-PNA hybrids. In accordance with the present invention, useful intercalators can be selected from the group consisting of ethidium bromide, diethidium bromide, acridine orange and SYBR Green. When employed in accordance with the present invention, the proteins generate a signal directly or indirectly. Such forms and manner of direct and indirect signal generation are as described elsewhere in this disclosure, particularly in several paragraphs above.
Related to the above described compositions are unique and useful processes. The present invention thus provides a process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of: a) providing: (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid anaiytes which may Enz-60 Elazar Rabbani et al., F~~~ng Date: Herewith Page 47 (New Patent Application) contain the nucleic acids of interest sought to be detected or quantified, wherein each of the nucleic acids of interest comprise at least one inherent universal detection target (UDT); and (iii) universal detection elements (UDE) which generates a signal directly or indirectly; b) hybridizing the library (ii) with the array of nucleic acids (i) to form hybrids if the nucleic acids of interest are present; c) contacting the UDEs with the UDTs to form a complex bound to the array; d) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. Many of these elements have been described previously in this disclosure, but at the risk of some redundancy, elaboration is now made. For example, the nucleic acid array can be selected from the group consisting of DNA, RNA and analogs thereof, the latter comprising PNA. Modifications to these nucleic acids and analogs can be usefully carried out to any one of the sugar, phosphate or base moieties. The solid support can be porous, e.g., polyacrylamide and agarose, or non-porous, e.g., glass or plastic. The solid support can also be transparent, translucent, opaque or reflective.
Nucleic acids are directly or indirectly fixed or immobilized to the solid support. Indirect fixation or immabilization to the solid support can be carried out by means of a chemical linker or linkage arm. As discussed elsewhere herein, the library of analytes can be derived from a biological source selected from the group consisting of organs, tissues and cells, or they may be from non-natural or more synthetic or man-made sources. Among biological analytes are those selected from the group consisting of genomic DNA, episornal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.
The inherent UDT used in the above process can be selected from the group consisting of 3' polyA segments, 5' caps, secondary structures, consensus sequences, and a combination of any of the foregoing. Such consensus sequences can be selected from the group consisting of signal sequences for polyA
addition, Enz-60 Elazar Rabbani et al., Fuing Date: Herewith Page 48 (New Patent Application) splicing elements, multicopy repeats, and a combination of any of the foregoing.
UDEs can be selected from the group consisting of nucleic acids, nucleic acid analogs, e.g., PNA, polypeptides, polysaccharides, synthetic polymers and a combination of any of the foregoing. UDEs generate a signal directly or indirectly.
Direct signal generation can be various and may be selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing. Indirect signal generation can also be _ various and may be selected from the group members consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a.
combination of any of the foregoing. When desired and employed in the process at hand, such an enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction. Those skilled in the art will readily appreciate that the above-described process can further comprise one or more washing steps.
This invention provides another such process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of a) providing: (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of the nucleic acids of interest comprise at least one inherent universal detection target (UDT); and (iii) universal detection elements (UDE) which generates a signal directly or indirectly; b) contacting the UDEs with the UDTs in the library of nucleic acid analytes to form one or more complexes; c) hybridizing the library of nucleic acid analyzes with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; d) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs Enz-60 Elazar Rabbani et al., f ,ding Date: Herewith Page 49 (New Patent Application) bound to the array. The nature and form of the nucleic acid array, modifications, solid support, direct/indirect fixation or immobilization, library of analytes, inherent UDT, UDE, direct/indirect signal generation, and the like, are as described elsewhere in this disclosure, including more particularly the last several paragraphs above. Furthermore, this process can comprise one or more conventional washing steps.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a . library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of the nucleic acids of interest comprise at least one non-inherent universal detection target (UDT), wherein the non-inherent UDT is attached to the nucleic acid analytes; and (iii) universal detection elements (UDE) which generate a signal directly or indirectly; b) hybridizing the library (ii) with the array of nucleic acids (i) to form hybrids if the nucleic acids of interest are present; c) contacting the UDEs with the UDTs to form a complex bound to the array; d) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. As described variously in this disclosure, the nature and form of the nucleic acid array, modifications to nucleic acid and nucleic acid analogs, the solid support, direct and indirectfixation/immobilization to the solid support, the library of analytes, direct and indirect signal generation, and the like, are as described elsewhere in this disclosure. Of particular mention are the non-inherent universal detection targets (UDTs) which can comprise homopolymeric sequences and heteropolymeric sequences. Also of particular mention are the universal detection elements (UDEs1 which can be selected from the group consisting of nucleic acids, nucleic acid analogs, e.g., PNA, and modified forms thereof. One or more washing steps can be included in this last process.
Enz-60 Elazar Rabbani et al., F~~~ng Date: Herewith Page 50 (New Patent Application) Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of such nucleic acids of interest comprise at least one non-inherent universal detection target (UDT), wherein the non-inherent UDTs are attached to the nucleic acid analytes; and (iii) universal detection elements (UDE) which generate a signal directly or indirectly; b) contacting the UDEs with the UDTs in the library of nucleic acid analytes to form _ ane or more complexes; c) hybridizing the library (ii) with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; d) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. Descriptions for the nucleic acid array, modifications, solid support, direct/indirect fixation or immobilization to the solid support, the library of analytes, the non-inherent universal detection targets (UDTs), the universal detection elements (UDEs), direct/indirect signal generation, inclusion of washing steps, and the like, are found elsewhere in this disclosure and are equally applicable to this last described process.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing ~i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) means for attaching one or more universal detection targets (UDT) to a nucleic acid; (iv) universal detection elements (UDE) which generates a signal directly or indirectly; b) attaching such UDTs (iii) to the library of nucleic acid analytes (ii); c) hybridizing the library (ii) with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; d) Enz-60 Elazar Rabbani et al., I-~~~ng Date: Herewith Page 51 (New Patent Application) contacting the UDEs with the UDTs to form a complex bound to the array; e) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. Many of these elements have been described already. These include the nucleic acid array, nucleic acid analogs, sugar, phosphate and base modifications, the solid support, direct/indirect fixation and immobilization to the solid support, the library of analytes, the universal detection elements, direct/indirect signal generation, inclusion of additional washing steps, and the like, have been described elsewhere above and below and are equally applicable to this last-mentioned process. Of special mention are attaching means which add homopolymeric sequences through various enzymes, e.g., poly A polymerase and terminal transferase. Other attaching means can be used for adding homopolymeric or heteropolymeric sequences, and these include enzymatic means and enzymes selected from DNA
ligase and RNA ligase.
Still another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) means for attaching one or mare universal detection targets (UDT) to a nucleic acid; (iv) universal detection elements (UDE) which generate a signal directly or indirectly; b) attaching the UDTs (iii) to the library of nucleic acid analytes (ii); c) contacting the UDEs with the UDTs in the library of nucleic acid analytes to form one or more complexes; d) hybridizing the library (ii) with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; e) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. As might be expected, the elements recited in this process have been described elsewhere in this disclosure and are equally applicable to this Enz-60 Elazar Rabbani et al., Fmng Date: Herewith Page 52 (New Patent Application) last described process. These previously described elements include the nucleic acid array, modifications, the solid support, direct/indirect fixation or immobilization to the solid support, the library of analytes, attaching means, UDE, directlindirect signal generation and the inclusion of washing steps.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) universal detection elements (UDEs) which bind to a domain formed by nucleic acid hybrids for complex formation and generate a signal directly or indirectly; b) hybridizing the library (ii) with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present, wherein any formed hybrids generate a domain for complex formation; c) contacting the UDEs with any hybrids to form a complex bound to the array; d) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array.
Descriptions for the nucleic acid array, nucleic acid analogs, e.g., PNA, modifications (sugar, base and phosphate moieties), the solid support, fixation/immobilization, the library of analytes, the domain for complex formation, direct/indirect signal generation from signaling proteins, washing steps, and the like, have already been given above and are equally applicable to this last mentioned process. Of special note is this process wherein the signaling entity is complexed to the domain for complex formation, such signaling entity being selected from proteins and intercalators. Such proteins can include nucleic acid binding proteins which bind preferentially to double-stranded nucleic acids, e.g., antibodies, particularly such antibodies which are specific for nucleic acid hybrids, e.g., DNA-DNA hybrids, DNA-RNA hybrids, RNA-RNA hybrids, DNA-PNA hybrids and RNA-PNA hybrids. Intercalators have also been previously described in this Enz-60 Elazar Rabbani et al., F",.~g Date: Herewith Page 53 (New Patent Application) disclosure and can be selected from ethidium bromide, diethidium bromide, acridine arange and SYBR Green.
Other compositions of matter are provided by this invention. One such composition comprises a library of first nucleic acid analyte copies, such first nucleic acid copies being hybridized to an array of nucleic acids, those nucleic acids being fixed or immobilized to a solid support, wherein such first nucleic acid copies comprise an inherent universal detection target (UDT) and a universal detection element (UDE) attached to the UDT, wherein the UDE generates a signal directly or indirectly to detect the presence or quantity of any analytes. The library of analytes, e.g., biological sources, and examples of such analytes, e.g., genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing, has been described above. Equally so, the nucleic acid array has been already described, including, for example, DNA, RNA and analogs thereof, e.g., PNA. Modifications to the nucleic acids and analogs (sugar, phosphate, base), features of the solid support (porous/non-porous, transparent, translucent, apaque, reflective), fixation/immobilization to the solid support, the inherent UDT, the UDE, direct/indirect signal generation from UDEs have been described above and apply equally to this last composition.
Another composition of matter comprises a library of first nucleic acid analyte copies, such first nucleic acid copies being hybridized to an array of nucleic acids, the nucleic acids being fixed or immobilized to a solid support, wherein such first nucleic acid copies comprise one or more non-inherent universal detection targets (UDTs) and one or more universal detection elements (UDEs) attached to the UDTs, wherein the UDEs generate a signal directly or indirectly to detect the presence or quantity of any analytes, and wherein the UDTs are either: (i) at the 5' ends of the first nucleic acid copies and not adjacent to an oligoT segment or sequence, or (ii) at the 3' ends of the first nucleic acid copies, or (iii) both (i) and (ii). The library of analytes, nucleic acid array, nucleic acid modifications, solid Enz-60 Elazar Rabbani et al., I-",ng Date: Herewith Page 54 (New Patent Application) support, fixation/immobilization to the solid support, non-inherent UDTs, e.g., heteropolymeric sequences, UDEs (e.g., nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, synthetic polymers, etc), direct/indirect signal generation from UDEs have already been described above and are applicable to this last described composition.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to the nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest _ sought to be detected or quantified, wherein each of such nucleic acids of interest comprise at least one inherent universal detection target (UDTI; (iii) universal detection elements (UDE) which generate a signal directly or indirectly; and (iv) polymerizing means for synthesizing nucleic acid copies of the nucleic acids of analytes; b) synthesizing one or more first nucleic acid copies which are complementary to all or part of the nucleic acid analytes and synthesizing sequences which are complementary to all or part of the UDT to form a complementary UDT; c) hybridizing such first nucleic acid copies with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; d) contacting the UDEs with the complementary UDTs of the first nucleic acid copies to form a complex bound to the array; e) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. Statements and descriptions for the nucleic acid array, modifications, solid support, fixation/irnmobilization, the library of analytes, inherent UDTs, e.g., consensus sequences, UDEs, direct/indirect signal generation from UDEs, have been given above and are equally applicable to this last process. Of special mention are the recited polymerizing means which can be selected from E. coli DNA Pol I, Klenow fragment of E, coli DNA Pol I, Bst DNA
polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA Polymerase, T4 Enz-60 Elazar Rabbani et al., F~~~ng Date: Herewith Page 55 (New Patent Application) DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV
reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.
Another embodiment provided by this invention is a process for detecting or quantifying more than one nucleic acid of interest in a Eibrary comprising the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of such nucleic acids of interest comprise at least one inherent _ universal detection target (UDT); (iii) universal detection elements (UDE) which generate a signal directly or indirectly; and (iv) polymerizing means for synthesizing nucleic acid copies of such nucleic acid analytes; b) synthesizing one or more first nucleic acid copies of such nucleic acid analytes; c) contacting the UDEs with the UDTs in the first nucleic acid copies to form one or more complexes; d) hybridizing such first nucleic acid copies with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; and e) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. The nucleic acid array, nucleic acid modifications, the solid support, fixation/immobilization (direct and indirect), the library of analytes, inherent UDTs, UDEs, signal generation from UDEs (direct/indirect), polymerizing means, have been described above. Such descriptions are equally applicable to this last process.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to the nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid; (iv) universal detection Enz-60 Elazar Rabbani et al., Firing Date: Herewith Page 56 (New Patent Application) elements (UDE) which generate a signal directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes; b) attaching the non-inherent UDTs to either the 3' ends of the nucleic acid analytes, the 5' ends of the first nucleic acid analytes, or both the 3' ends and the 5' ends of the nucleic acid analytes; c) synthesizing one or more first nucleic acid copies of the nucleic acid analytes; d) hybridizing the first nucleic acid copies with the array of nucleic acids (i) to form hybrids if such nucleic acids of interest are present; e) contacting the UDEs with the UDTs of the first nucleic acid copies to form a complex bound to the array; and f) detecting or quantifying the more than one - nucleic acid of interest by detecting or measuring the amount of .signal generated from UDEs bound to the array. See many of the preceding paragraphs for descriptions and characteristics of the nucleic acid array, modifications, the solid support, fixation/immobilization, the library of analytes, attaching means, UDEs, direct/indirect signal generation from UDEs, polymerizing means, and the like.
Yet another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to the nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid; (iv) universal detection elements (UDE) which generate a signal directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes; b) attaching such non-inherent UDTs to either the 3' ends of the nucleic acid analytes, the 5' ends of the first nucleic acid analytes, or both the 3' ends and the 5' ends of the nucleic acid analytes; c) synthesizing one or more first nucleic acid copies of the nucleic acid analytes; d) contacting the UDEs with the UDTs of the first nucleic acid copies to form complexes; e) hybridizing the first nucleic acid copies with the array of nucleic acids (i) to form hybrids if any nucleic acids of interest are present;
Enz-60 Elazar Rabbani et al., Fmng Date: Herewith Page 57 (New Patent Application) f) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. The nucleic acid array, modifications, the solid support, direct/indirect fixation/immobilization, the library of analytes, attachment means, UDEs, signal generation from UDEs, direct/indirect signal generation, polymerizing means, and the like, have already been described. Such descriptions are equally applicable to this last-described process.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or _ immobilized nucleic acids identical in part or whole to such nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid; (iv) universal detection elements (UDE) which generate a signal directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes; b) synthesizing one or more first nucleic acid copies of the nucleic acid analytes; c) attaching the non-inherent IJDTs to either the 3' ends of the first nucleic acid copies, the 5' ends of the first nucleic acid copies, or both the 3' ends and the 5' ends of the first nucleic acid copies; d) hybridizing the first nucleic acid copies with the array of nucleic acids Ii) to form hybrids if any nucleic acids of interest are present; e) contacting the UDEs with the UDTs of the first nucleic acid copies to form a complex bound to the array; and f) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. Descriptions for the above-recited elements have been given above and are equally applicable to this last process.
Still another process provided by this invention is for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a1 providing (i) an array of fixed or immobilized nucleic acids identical in part or whole Enz-60 Elazar Rabbani et al., Firing Date: Herewith Page 58 (New Patent Application) to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid; (iv) universal detection elements (UDE) which generate a signal directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes; b) synthesizing one or more first nucleic acid copies of the nucleic acid analytes; c) attaching the non-inherent UDTs to either the

3' ends of the first nucleic acid copies, the 5' ends of the first nucleic acid copies, ar both the 3' ends and the 5' ends of the first nucleic acid copies; d.) contacting - the UDEs with the UDTs of the first nucleic acid copies to form a complex;
e) hybridizing the first nucleic acid copies with the array of nucleic acids (i) to form hybrids if any nucleic acids of interest are present; and f) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. These elements and subelements have been described elsewhere in this disclosure. Such descriptions apply to this last process.
Yet another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids complementary to the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) universal detection elements (UDEs) which bind to a domain for complex formation formed by nucleic acid hybrids and generate a signal directly or indirectly; and (iv) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes; b) synthesizing one or more nucleic acid copies of the nucleic acid analytes; c) hybridizing the first nucleic acid copies with the array of nucleic acids (i) to form hybrids if any nucleic acids of interest are present, wherein any formed hybrids generate a domain for complex formation;
d) contacting the UDEs with the hybrids to form a complex bound to the array; and e) Enz-60 Elazar Rabbani et al., F~~~ng Date: Herewith Page 59 (New Patent Application) detecting or quantifying the more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to the array. The above-recited elements and subelements and variations thereof are described elsewhere in this disclosure and are equally applicable to this just-mentioned process.
One aspect of the present invention discloses methods that eliminate the necessity for enzymatic incorporation of labeled nucleotides by an end user.
In this particular aspect, common or conserved features present in a diverse population of nucleic acid analytes are used to assay the extent of hybridization of the analytes _ to discrete target elements in an array format. These common or conserved features are Universal Detection Targets (UDTs) which can provide signal generation by binding of Universal Detection Elements (UDEs).
Examples of UDTs that may be inherently present in a population of diverse nucleic acid analytes can comprise but not be limited to 3' poly A segments, 5' caps, secondary structures and consensus sequences. Examples of consensus sites that might find use in the present invention can comprise but not be limited to signal sites for poly A addition, splicing elements and multicopy repeats such as Alu sequences.
UDEs may be directly or indirectly labeled. Examples of directly labels can comprise but not be limited to any members of a group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.
Examples of indirect labels can comprise but not be limited to any members of a group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing. Among such enzymes are any enzymes which catalyze reactions selected from the group Enz-60 Elazar Rabbani et al., Fnmg Date: Herewith Page 60 (New Patent Application) consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.
RNA and DNA polymerases sometimes have difficulty in accepting labeled nucleotides as substrates for polymerization. In prior art, this shortcoming can result in the production of a labeled library that consists of short strands with few signal generating entities. Limitations caused by such inefficient incorporation can be partially compensated for by increasing the amount of labeled precursors in the reaction mixtures. However, this method achieves only a moderate improvement and entails a higher cost and waste of labeled reagents. In contrast, this particular aspect of the present invention discloses means by which diverse nucleic acids in a library can be hybridized in an array format in their native form without the need of any manipulations or modifications and then be detected by the presence of UDTs bound to the array.
An illustrative depiction of this process is given in Figure 1. Although there are multiple unique species of mRNA that can make up a diverse population of nucleic acids in a sample, the common elements that are shared by these nucleic acids can be used as UDTs. Hybridization of the mRNA to an array permits the localization of individual species to discrete locations on the array. The determination of the amount of sample that is bound to each locus of an array is then carried out by detection of the amount of UDT present at each locus by binding of the appropriate UDE. Thus, in Figure 1, locus 1 and 3 would be capable of generating an amount of signal that would be proportionate to the amount of mRNA bound to each of those sites. On the other hand there would little or no signal generation from locus 2 since there was little or no mRNA bound to that site.
A single labeled species of mostly or completely poly T or U could be used as a UDE to quantify the amount of poly A tails of the various species of eucaryotic mRNA in Figure 1. In this way, a single universal species of labeled material is Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 61 (New Patent Application) synthesized for use as a UDE thereby providing an inexpensive and efficient means of indirectly labeling the RNA molecules being quantified.
A nucleic acid UDE can be prepared either chemically or enzymatically. For example, oligonucleotide synthesizers are commercially available that can produce a UDE consisting of labeled poly T/U sequences for detection of the poly A UDT
described above. Both the amount and placement of labeled moieties can be tightly controlled by this method. Also, since this is a homopolymeric product, probes that are shorter by one or more bases will still be effective such that the net yield of usable product will be higher than one that requires a discrete specific sequence. On the other hand, methods of synthesizing such sequences enzymatically are also well known to those versed in the art. Commonly, a tetramer of dT is used as a primer for addition of poly T or poly U by terminal transferase. Each base can be modified to be capable of signal generation or a mixture of labeled and unlabeled bases can be used. Although A Poly A UDT has been described in the example above, when different sequences are used as UDTs, the synthesis of the coresponding UDEs can be carried out by the same chemical and enzymatic methodologies described above. It is also contemplated that analogues of DNA can also be used to synthesize the UDEs. For instance, instead of using DNA, labeled RNA or PNA /peptide nucleic acids) may also be used.
Detection and quantification of the amount of UDTs bound to particular loci can also be carried out by the use of an antibody acting as a UDE. Examples of antibody specificities that are useful for UDEs can comprise but not be limited to recognition of the cap element at the 5' end of mature mRNAs or the homopolymeric poly A sequence. Furthermore, hybridization between nucleic acids is an event that in and of itself is capable of generating a UDT that can be recognized by antibody UDEs. For example, when a library of diverse RNA
species are bound to an array, the RNA, DNA or PNA target elements in the array will generate RNA/RNA, RNA/DNA or RNA/PNA hybrids at each of the loci that has Enz-60 Elazar Rabbani et al., Fn~og Date: Herewith Page 62 (New Patent Application) homology with the particular RNA species being quantified. Although each of the sites has a discrete sequence, universal detection and quantification can be carried out by antibodies that recognize the change in physical structure produced by such hybridization events. Alternatively, the hybridization between a UDE and the complementary UDT of a nucleic acid bound to the target elements of the array can be detected by an appropriate antibody. The antibodies that are specific for the UDEs described above can be labeled themselves or secondary labeled antibodies can be used to enhance the signal.
If only a single library of mRNA is being analyzed, binding of a UDE to a UDT
_ may take place before or after hybridization of the RNA to an array of detection probes. The particular order of events will depend upon the nature and stability of the binding partners. When analytes from two libraries are intended to be compared simultaneously, binding of each UDE to a binding partner is preferably carried out prior to hybridization of the RNA to an array of target elements such that each library is differentially labeled. Although comparisons are typically carried out between two libraries, any number of comparisons can be made simultaneously as long as each library is capable of generating a signal that can be distinguished from the other libraries. On the other hand, rather than simultaneous hybridization and detection, the arrays can be used in a parallel or sequential fashion. In this format, hybridization and detection is carried out separately for each library and the analysis of the results is compared afterwards relative to normalized controls of steady state genes.
In another aspect of the present invention, UDTs or UDEs are artificially incorporated into the diverse nucleic acids of the library. Enzymes that find particular use with RNA analytes may comprise but not be limited to Poly A
polymerise which specifically adds Adenine ribonucleotides to the 3' end of RNA
and RNA ligase which can add an oligonucleotide or polynucleotide to either the 5' or 3' end of an RNA analyte. By these means, either homopolymeric or unique Enz-60 Elazar Rabbani et al., Fn~og Date: Herewith Page 63 (New Patent Application?
sequences can be added to serve as UDTs or UDEs. Enzymes that find particular use with DNA analytes may comprise but not be limited to Terminal Transferase for addition to 3' ends and DNA ligase for addition to either 3' or 5' ends. The sequences that are introduced into the nucleic acid analytes can be labeled during synthesis or addition of a UDE or conversely unlabeled UDTs can be synthesized or added that are detected later by corresponding labeled UDEs. This aspect enjoys special utility when unspliced RNA, snRNA, or rRNA are used as analytes since they may be lacking inherent elements that are present in mRNA that have previously cited as being useful as UDTs. This aspect of the present invention will _ also find use with procaryotic mRNA since the poly A additions, 5' caps and splicing elements which have been previously cited as potential UDTs of mRNA
are intrinsically lacking in procaryotes.
This particular aspect of the present invention may also be used in conjunction with fragmentation processes. For instance, mRNA molecules from eucaryotic organisms can be very large even after processing events have taken place. This size factor can hinder hybridization or allow scissions between the segment used for binding to a target element in the array and the UDT that is being used for signal generation. Additionally, a fragmentation step may also reduce the amount of secondary structure present in RNA. Therefore, in this aspect of the present invention, RNA can be fragmented into smaller sized pieces either by physical or enzymatic followed by addition of sequences that can act as UDTs or UDEs. Examples of physical means for fragmentation of nucleic acids can include but not be limited to shearing or alkali treatment. Examples of enzymatic means can include but not be limited to a partial nuclease or RNase digestion.
In addition, DNA from most sources will also be extremely large in its native form. DNA anaiytes may also be fragmented by suitable physical or enzymatic means. A particularly useful enzymatic means would be the use of restriction enzymes where the nature of the recognition sequence for the restriction enzyme Enz-60 Elazar Rabbani et al., F~"~ig Date: Herewith Page 64 (New Patent Application) will determine the average size of the fragments. Also, although most restriction enzymes require double-stranded DNA as templates, some enzymes such as Hha I, Hin P1 I and Mnl I cleave single-stranded DNA efficiently (2000-2001 catalog, New England BioLabs, Beverly, MA, p214). By this fragmentation method a single analyte molecule is converted into multiple subfragments that can each have their own artificially introduced UDT or UDE. An exemplary illustration of this particular aspect of the present invention is included in Figure 2.
In another aspect of the present invention, the diverse nucleic acids in a library are used as templates for synthesis of complementary nucleic acid copies instead of using the analytes directly for array analysis. The analyte templates may have intrinsic UDTs present or they may have UDTs artificially incorporated by the means cited earlier. On the other hand, the UDTs do not have to be present in the analyte templates and incorporation of artificial UDTs can take place either during or after synthesis of nucleic acid copies. Examples of enzymes that may be used for making copies of DNA templates can comprise but not be limited to DNA
polymerases for synthesis of DNA copies and RNA polymerases for the synthesis of RNA copies. Examples of DNA polymerases that may have use in the present invention for synthesis of DNA copies from DNA templates can include but not be limited to E.coli DNA Pol I, the Klenow fragment of E, coli DNA Pol I, Bst DNA
polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, ALV Reverse Transcriptase, RSV Reverse Transcriptase, HIV-1 Reverse Transcriptase, HIV-2 Reverse Transcriptase, Sensiscript, Omniscript and various mutated or otherwise altered forms of the foregoing. Examples of RNA polymerases that may have use in the present invention for synthesis of RNA copies from DNA templates can include but not be limited to bacteriophage T3 RNA polymerase, bacteriophage T7 RNA polymerase and bacteriophage SP6 RNA polymerase. Examples of enzymes that may have use in the present invention for making DNA copies of RNA templates can comprise but Enz-60 Elazar Rabbani et al., r~nng Date: Herewith Page 65 (New Patent Application) not be limited to ALV Reverse Transcriptase, RSV Reverse Transcriptase, HIV-1 Reverse Transcriptase, HIV-2 Reverse Transcriptase, Sensiscript, Omniscript, Bst DNA polymerise, Bca DNA polymerise, Tth DNA polymerise and various mutated or otherwise altered forms of the foregoing.
Examples of enzymes that may have use in the present invention for making RNA copies of RNA templates can comprise but not be RNA dependent RNA
polymerises (Koonin, 1991 J. Gen Virol. 72; 2197-2206, incorporated herein by reference) .
Efficient synthesis of complementary copies of analyte templates require the presence of a promoter for efficient synthesis by DNA dependent RNA
polymerises while the other cited exemplary enzymes require primers. Incorporation of a UDT
into a DNA analyte that will be transcribed by a DNA dependent RNA polymerise can comprise but not be limited to ligation of a UDT sequence and a promoter sequence by the action of DNA ligase. This process is depicted below:
DNA analyte + UDT--Promoter - DNA Analyte--UDT--Promoter Transcription of this construct would then be capable of production of RNA
with the structure: 3' analyte--UDT 5':
One means of carrying out this particular aspect of the present invention is digestion of a library of diverse double-stranded DNA analytes by a restriction enzyme followed by ligation of a double-stranded DNA segment comprising an RNA
promoter sequence. Subsequent transcription of the transcription units can synthesize either labeled or unlabeled transcripts. The unlabeled transcripts can be detected by the presence of either inherent or synthetically added UDTs.
When primers are used for synthesis of complementary copies of analyte templates, the primers can comprise random sequences or selected sequences for Enz-60 Elazar Rabbani et al., r ~nng Date: Herewith Page 66 (New Patent Application) binding to the analyte templates. Random primers that have commonly been used for priming events have ranged from hexamers to dodecamers. Selected sequences that are useful as primers can be complementary to inherent sequences or to non-inherent sequences that have been introduced into the analyte templates.
Examples of inherent sequences can include but not be limited to consensus sequences or homopolymeric sequences. Consensus sequences can be derived from elements that are retained in a large portion of the population being studied.
Examples of these could comprise but not be limited to poly A addition sites, splicing elements and multicopy repeats such as Alu sequences. An example of inherent homopolymeric sequences used for primer binding can be the poly A
tail that is intrinsic to mature mRNA in eucaryotes. Non-inherent homopolymeric or unique sequences that can be used for primer binding may be introduced into RNA
templates by means that can include but not be limited to poly A polymerase or RNA ligase. Non-inherent homopolymeric or unique sequences that can be used for primer binding may be introduced into DNA templates by means that can include but not be limited to Terminal Transferase and DNA ligase. The artifjcial binding sites can be introduced into intact nucleic acid templates or fragmentation processes may be carried out as described previously.
When homopolymeric or conserved sequences are used as primer binding sites, the library can be subdivided by the use' of primers that have been synthesized with 1 or more additional discrete bases at the 3' end. For example, an oligonucleotide primer that has the formula 5'-T"dC-3' would preferentially prime mRNAs whose last base was a G before the poly A tail rather than priming the entire population of mRNA's with poly A tails. The same principle would also hold true when either 5'-T"dG-3' or 5'-T"dA-3' primers are used. This would provide three separate sub-populations of copies of the original mRNA population that in t'oto should encompass the entire RNA population with poly A tails. This population could be further divided by inclusion of a 2"d discrete base at the 3' end Enz-60 Elazar Rabbani et al., r ~nng Date: Herewith Page 67 (New Patent Application of the primers. In this ease, oligonucleotides would have either dC, dG, dA or dT
as the last base at the 3' end and dC, dG or dA in the penultimate position and the remaining portion comprising a poly T segment. This would create the potential for ~ 2 separate pools from the original population. Further provision of discrete bases at the 3'd nucleotide position from the 3' end would provide a separation into different subpopulations if desired and so on.
The use of subpopulations may have utility in providing RNA with lower complexity thereby simplifying analysis later on. In addition, the use of discrete bases at the 3' end would limit the size of poly T tails at the end of the cDNA
copies since significant amounts of priming events will only take place at the junction of the poly A addition site. This may reduce background hybridization caused by extensive polyT or PolyA tracts. Also it may increase yields of labeled products by decreasing stalling or premature terminations caused by long homopolymeric tracts. On the other hand, the use of a mixture of oligo T
primers with discrete bases at the 3' end would be similar to a completely homopolymeric oligo T primer in being able to synthesize a complete representation of the original analyte sequences while at the same retaining the ability to constrain the size of homopolymeric tails.
In this particular aspect of the present invention, the cDNA molecules synthesized from the pool of RNA templates also comprise UDTs or UDEs. As described previously, these UDTs can be inherently present or they may be non-inherent sequences that are artificially incorporated during synthesis of cDNA.
When an analyte has a nucleic acid sequence that can be used as a UDT, synthesis of the complementary copy creates a sequence that can also be used as a UDT.
For example, the poly A sequence at the 3' end of eucaryotic mRNA was previously described as a potential UDT. When this mRNA is used as a template by extension of a poly T primer with or without additional bases, the poly T
segment of the cDNA copy can function as a UDT. The destruction or separation of the Enz-60 Elazar Rabbani et al., r ,~~ng Date: Herewith Page 68 (New Patent Application) RNA templates from the cDNA would allow the poly T at the 5' end of the cDNA
to act as a UDT by binding of a labeled poly A UDE. UDTs or UDEs can also be incorporated into cDNA copies by inclusion of nucleic acid segments that don't participate in primer binding into the 5' tails of either random, homopolymeric, or specific sequence primers. The particular sequence of the additional nucleic acid segments used as UDTs are of arbitrary nature since they aren't needed for primer binding. As such, the choice of sequence for these UDTs can range in complexity from homopolymeric sequences to specific unique sequences. Their nature is also arbitrary, and either the primer or the UDT can comprise PNA's or other nucleic acid homologues. In addition, they may be other polymeric entities besides nucleic acids that provide recognition for UDEs.
Since the nature of the UDT or UDE can be selected by the user, the present invention allows simple differentiation between libraries that are being compared.
For instance, one population that is being studied can be extended by homopolymeric or random primers and hybridized with a UDE labeled with Cy 3. A
second population that is being compared can be extended by homopolymeric or random primers and hybridized with UDEs that have Cy 5 incorporated into them.
The other end of the cDNA is also available for use with UDEs. For example, after synthesis of cDNA copies by reverse transcriptase, the 3' ends can be extended further by the non-template directed addition of nucleotides by Terminal Transferase. An illustration of this particular aspect of the present invention is included in Figure 3.
Detection of the presence of UDTs or UDEs in the library or libraries of various nucleic acids can be carried out by any of the means that have been described previously for UDTs. ff only a single library is being analyzed, binding of a probe or antibody to a 5' or 3' UDT or UDE may take place before or after hybridization of nucleic acids to the detection elements of the array. The particular order of events will depend upon the nature and stability of the binding partners.
Enz-60 Elazar Rabbani et al., r ~~~ng Date: Herewith Page 69 (New Patent Application) On the other hand, when each population incorporates a different UDT or UDE, binding of labeled moieties to the UDTs can take place either before or after hybridization of the copies of the analyte to an array. However, as described previously, the same UDT or UDE can be used for each population if parallel or sequential hybridizations are carried out.
It is also contemplated that the various aspects of the present invention can be used to augment rather than substitute for other previously disclosed methods.
For instance, signal can be generated in cDNA copies by a labeled primer being extended in the presence of labeled nucleotides. The signal generated by such a _ method would be a summation of the signal generated by the original primer and whatever labeled nucleotides were incorporated during strand extension. Thus, a combination of methodologies would generate a signal that would be higher than the amount that would be achieved by either method alone. In addition to a pre-labeled primer, the other methods that are disclosed in the present invention can also be used in various combinations.
There may be situations where amplification of sequences in a sample is advantageous. Therefore, in another aspect of the present invention, multiple cycles of synthesis can be carried out to generate linear amplification of a library of diverse nucleic acid sequences. In the first step of this particular aspect of the present invention, the entire population or a subset of the population of nucleic acids analytes is used to synthesize 1 st strand nucleic acid copies. Whether the initial analyte is DNA or RNA, in the context of the present invention, this product is considered to be a cNA since it represents a nucleic acid copy of the analyte.
Synthesis of the 1" strand nucleic acid copies can be carried out as described previously by using discrete primes, random primers, homopolymers, or homopolymers with one or more discrete bases at their 3' ends. In this particular embodiment of the present invention, priming with homopolymers with one or more discrete bases at their 3' ends may also increase the efficiency of amplification Enz-60 Elazar Rabbani et al., . ~~ing Date: Herewith Page 70 (New Patent Application) since resources such as primers and substrates will be directed only towards amplification of a discrete subpopulation derived from the 1't cNA synthesis reaction.
For linear amplification, a prirner binding site on a nucleic acid analyte is used multiple times by separation of a 1 S' cNA copy from its template followed by reinitiation of a new 1 °t cNA copy. Separation can be carried out by exposure of the reaction mix to high temperature. If the enzyme used for nucleic acid synthesis is Taq polymerase, Tth polymerase or some other heat stable polymerase the multiple reactions can be carried out by thermocycling of the reaction without the _ addition of any other reactions. On the other hand, if high denaturation temperatures are used in conjunction with enzymes that are heat labile, for instance Bst DNA polymerase, Klenow fragment of Pol I or MuLV Reverse 'Transcriptase, irreversible heat inactivation of the enzyme takes place and the enzyme has to be replenished far further rounds of cNA synthesis.
Alternatively, methods have been disclosed by Fuller in U.S. Patent No. 5,432065 and by ~akobashvill and Lapidot, 1999 (Nucleic Acids Research 27; 1566-1568) for reagents that allow low temperature denaturation of nucleic acids for use with PCR, both of which methods are incorporated by reference. Furthermore, Winhoven and Rossau have disclosed in PCT Application WO 98/45474 (also incorporated by reference) that temperature manipulation can be avoided completely by electrically controlled manipulation of divalent ion levels.
Thus by these methods even thermo-labile enzymes can carry out multiple cycles of synthesis for linear amplification. Both above-cited patent documents and the above-cited publication are incorporated herein by reference.
Amplification is a significant aspect of this invention. Several compositions and processes are devoted and directed to amplification. For example, provided herein is a composition of matter comprising a library of double-stranded nucleic acids substantially incapable of in vivo replication and free of non-inherent ~nz-60 Elazar Rabbani et al., . .,mg Date: Herewith Page 71 (New Patent Applications homopolymeric sequences, the nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample, wherein the double-stranded nucleic acids comprise at least one inherent universal detection target (UDTS proximate to one end of the double strand and at least one non-inherent production center proximate to the other end of the double strand. The sample from which the inherent sequences of the library are obtained can comprise biological sources, e.g., organs, tissues and cells. As described elsewhere herein, the library of nucleic acids can be derived from genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of . the foregoing. Inherent UDTs can be selected from the group consisting of 3' polyA segments, consensus sequences, or both. As already described above, consensus sequences can be selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing. Of special mention is the production center which can be selected from the group consisting of primer binding sites, RNA
promoters, or a combination of both. Such RNA promoters can comprise phage promoters, e.g.. T3, T7 and SP6.
Another composition of matter for amplification purposes comprises a library of double-stranded nucleic acids substantially incapable of in vivo replication, such nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample, wherein the double-stranded nucleic acids comprise at least four 441 non-inherent nucleotides proximate to one end of the double strand and a non-inherent production center proximate to the other end of the double strand. Descriptions for such elements, i.e., the sample, the library of nucleic acids, inherent UDTs, non-inherent nucleotides, non-inherent production centers, e.g., RNA promoters, e.g., phage promoters (T3, and SP6s are given elsewhere in this disclosure and are equally applicable to this last composition.
Enz-60 Elazar Rabbani et al., . ,nng Date: Herewith Page 72 (New Patent Application) Another composition of matter for amplification comprises a library of double-stranded nucleic acids fixed to a solid support, those nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample and the nucleic acids further comprising at least one first sequence segment of non-inherent nucleotides proximate to one end of the double strand and at least one second sequence segment proximate to the other end of the double strand, the second sequence segment comprising at least one production center. Of special mention is the use of beads as the solid support, particularly beads and magnetic beads. Other _ elements, such as the sample and biological sources, the library of nucleic acids, inherent UDTs, non-inherent production centers, have already been described.
Yet another amplification type composition of matter comprises a library of double-stranded nucleic acids attached to a solid support, the nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample, wherein the double-stranded nucleic acids comprise at least one inherent universal detection target (UDT) proximate to one end of the double strand and at least one non-inherent production center proximate to the other end of the double strand. The elements and subelements (solid support, beads, magnetic beads, sample, library of nucleic acids, inherent UDTs, consensus sequences, production centers, RNA promoters, phage promoters, e.g., T3, T7 and SP6, have been described above.
Among useful processes for detecting or quantifying more than one nucleic acid of interest in a library, one such process of the present invention comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, the polymerizing means comprising Enz-60 Elazar Rabbani et al., ~ ,nng Date: Herewith Page 73 (New Patent Application) a first set of primers and a second set of primers, wherein the second set of primers comprises at least two segments, the first segment at the 3' end comprising random sequences, and the second segment comprising at least one production center; (iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions; b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) contacting the extended first copies with the second set of primers to form more than one second bound entity;
. e) extending the bound second set of primers by means of template sequences provided by the extended first copies to form more than one complex comprising extended first copies and extended second set of primers; f) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; g) hybridizing any nucleic acid copies formed in step f) to the array of nucleic acids provided in step a) (i); and h) detecting or quantifying any of the hybridized copies obtained in step g). Elements recited in the process just above and their subelements have already been described in this disclosure. Of special mention is the first set of primers which are complementary to inherent UDTs. Further mention should be made that the hybridized nucleic acids can comprise one or more signaling entities attached or incorporated thereto. As described variously above, signal detection can be carried aut directly or indirectly. Mention is also made that the process can further comprise the step of separating the first copies obtained from step c) from their templates and repeating step b). Other steps can also be included such as the step of separating the extended second set of primers obtained from. step f) from their templates and repeating step e). Step g) can also be carried out repeatedly, a feature provided by this invention and this last-described process. Further, means for synthesizing nucleic acid copies under isothermal or isostatic conditions is Enz-60 Elazar Rabbani et al., twing Date: Herewith Page 74 (New Patent Application) carried out by one or more members selected from the group consisting of RNA
transcription, strand displacement amplification and secondary structure amplification. These are all contemplated for use of this process.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid _ analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the first set of primers comprise at least one production center; and fiv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions; b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) extending the first copies by means of at least four (4) or more non-inherent homopolymeric nucleotides; e) contacting the extended first copies with the second set of primers to form more than one second bound entity; f) extending the bound second set of primers by means of template sequences provided by the extended first copies to form more than one complex comprising extended first copies and extended second set of primers;
g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions;
h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step a) Ii); and i) detecting or quantifying any of the hybridized copies obtained in step h). Of special mention is the use or addition of terminal transferase in or after extending step d) wherein the four or more non-inherent homopolymeric nucleotides are themselves added. Elements and subelements of Enz-60 Elazar Rabbani et al., r ~;mg Date: Herewith Page 75 (New Patent Application) this process are described above. Special mention is made of certain aspects of this process. For example, means for synthesizing nucleic acid copies under isothermal or isostatic conditions can be carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification. Moreover, the step of separating the first copies obtained from step c) from their templates and repeating step b) can be added to this process. Moreover, the extended second set of primers obtained from step f) can be separated from their templates and then step e) can be repeated as necessary or desired. In fact, step g) can be repeated as _ often as desired or deemed necessary.
A process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the first set comprises at least one production center; (iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of the second set of primers; andlv) means for ligating the set of oligonucleotides or polynucleotides (iv); b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) ligating the set of oligonucleotides or polynucleotides a) (iv) to the 3' end of the first copies formed in step c) to form more than one ligated product; e) contacting the ligated product with the second set of primers to form more than one second bound entity; f) extending the bound second set of primers by means of template sequences Enz-60 ~lazar Rabbani et al., tming Date: Herewith Page 76 (New Patent Application) provided by the ligated products formed in step d) to form more than one complex comprising the ligated products and the extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions;
h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of the hybridized copies obtained in step h). Aspects of this process, including the nucleic acid array, modifications, solid support, fixation/immobilization, nucleic acid analytes, UDTs, production centers, signal generation, polymerizing means, additional steps and repeating steps, synthesizing means, and so forth, have been described above and apply equally to this last-mentioned process. Of special mention are the above-recited ligating means which can comprise, for example, T4 DNA ligase.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the second set comprises at least one production center;
(iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of the second set of primers; and (v) means for ligating the set of oligonucieotides or polynucleotides (iv); b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity;
c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) ligating the set of oligonucleotides or polynucleotides a) (iv) to the 3' end of the first copies formed in step c) to form more than one ligated product; e) contacting the ligated Enz-60 Elazar Rabbani et al., r,~~ng Date: Herewith Page 77 (New Patent Application) product with the second set of primers to form more than one second bound entity; f) extending the bound second set of primers by means of template Sequences provided by the ligated products formed in step d) to form more than one complex comprising the ligated products and the extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of the hybridized copies obtained in step hl. Each of the above-recited elements in this _ process have been described elsewhere in this disclosure. Such descriptions are equally applicable to this process. Of special mention is the process wherein the first set of primers comprise one ar more sequences which are complementary to inherent UDTs. The hybridized nucleic acid copies can further comprise one or more signaling entities attached or incorporate thereto. If so, previously described embodiments for signal generation and detection, e.g., direct and indirect generation and detection, are applicable to this process. As described previously for other similar processes, additional steps can be carried out. For example, the step of separating the first copies obtained from step c) from their templates and then repeating step b) can be carried out. A further step of separating the extended second set of primers obtained from step f) from their templates and then repeating step e) can be carried out. Also, step g) can be carried out repeatedly.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers, a second set Enz-60 Elazar Rabbani et al., . .,~ng Date: Herewith Page 78 (New Patent Application) of primers and a third set of primers wherein the third set comprises at least one production center; and b) contacting the library of nucleic acid analytes with the first set of primers to form a first set of bound primers; c) extending the first set of bound primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) contacting the extended first copies with the second set of primers to form a second set of bound primers; e) extending the second set of bound primers by means of template sequences provided by the extended first copies to form second copies of the nucleic acid analytes; f) contacting the second copies with the third set of primers to form more than one third bound entity to form a third set of bound primers; g) extending the third set of bound primers by means of template sequences provided by the extended second set of primers to form a hybrid comprising a second copy, a third copy and at least one production center; h) synthesizing from the production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; i) hybridizing the nucleic acid copies formed in step i) to the array of nucleic acids provided in step a) (i); and j) detecting or quantifying any of the hybridized copies obtained in step i). Elements recited in this process and variations and subelements are as described elsewhere in this disclosure. Of special mention is the use of random primers as the second set of primers.
Furthermore, the second set of primers can be complementary to the primer binding site where the process comprises an additional step c') of including a primer binding site after carrying out step c). The primer binding site can be added by means of T4 DNA ligase or terminal transferase. Other aspects or variations of this process can be made or carried out. The further step of separating the extended second set of primers obtained from step f) from their templates and then repeating step e) can be made. Step g) can also be carried out repeatedly. An additional step f') of separating the extended second set of primers obtained in step e) can be carried out. Also, the step of separating the first copies obtained from step c) from Enz-60 Elazar Rabbani et al., ~ "mg Date: Herewith Page 79 (New Patent Application) their templates and then repeating step b) can be carried out. Further, the step of separating the extended second set of primers obtained from step f) from their templates and then repeating step e) can be carried out. Step g) can also be carried out repeatedly. In another variation of this process, the second set of primers can comprise at least one production center which differs in nucleotide sequence from the production center in the third set of primers.
Still another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences _ of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the first set of primers are fixed or immobilized to a solid support, and wherein the second set of primers comprises at least two segments, the first segment at the 3' end comprising random sequences, and the second segment comprising at least one production center; (iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions; b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) contacting the extended first copies with the second set of primers to form more than one second bound entity; e) extending the bound second set of primers by means of template sequences provided by the extended first copies to form more than one complex comprising extended first copies and extended second set of primers; f) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; g) hybridizing the nucleic acid copies formed in step f) to the array of Enz-60 Elazar Rabbani et al., . ,ding Date: Herewith Page 80 (New Patent Application) nucleic acids provided in step a) ii); and h) detecting or quantifying any of the hybridized copies obtained in step g). The above-recited elements and variations and subelements thereof have been described elsewhere and previously in this disclosure. Those descriptions apply equally to this process.
Another significant process worth discussion is one for detecting or quantifying more than one nucleic acid of interest in a library. This process comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the first set of primers are fixed or immobilized to a solid support, and wherein the first set of primers comprise at least one production center; and (iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions; b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) extending the first copies by means of at least four (4) or more non-inherent homopolymeric nucleotides; e) contacting the extended first copies with the second set of primers to form more than one second bound entity;
f) extending the bound second set of primers by means of template sequences provided by the extended first copies to form more than one complex comprising extended first copies and extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions; h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of the hybridized copies obtained in step Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 81 (New Patent Application) h). The elements recited above in this process and variations and subelements are described elsewhere in this disclosure. Those descriptions apply to this process.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
~iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers, wherein the first set of primers are fixed or immobilized to a solid support, and wherein the first set comprises at least one production center;
(iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of the second set of primers; and (v) means for ligating the set of oligonucleotides or polynucleotides (iv); b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) ligating the set of oligonucleotides or polynucleotides a) (iv) to the 3' end of the first copies formed in step c) to form more than one ligated product; e) contacting the ligated product with the second set of primers to form more than one second bound entity; f1 extending the bound second set of primers by means of template sequences provided by the ligated products formed in step d) to form more than one complex comprising the ligated products and the extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions;
h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of the hybridized copies obtained in step h). Descriptions for any of the above-recited elements in this Enz-60 Elazar Rabbani et al., t ..,ng Date: Herewith Page 82 (New Patent Application) process are given elsewhere in this disclosure, and need not be repeated except to say that such are equally applicable to this process.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second _ set of primers, wherein the first set of primers are fixed or immobilized to a solid support, and wherein the second set comprises at least one production center;
(iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of the second set of primers; and (v) means for ligating the set of oligonucleotides or polynucleotides (iv); b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) ligating the set of oligonucleotides or polynucleotides a) (iv) to the 3' end of the first copies formed in step c) to form more than one ligated product; e) contacting the ligated product with the second set of primers to form more than one second bound entity; f) extending the bound second set of primers by means of template sequences provided by the ligated products formed in step d1 to form more than one complex comprising the ligated products and the extended second set of primers; g) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions;
h) hybridizing the nucleic acid copies formed in step g) to the array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of the hybridized copies Enz-60 Elazar Rabbani et al., F~,.~og Date: Herewith Page 83 (New Patent Application) obtained in step h1. For a description of the elements recited in this process, refer to any of the several preceding paragraphs.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers, a second set of primers and ~a third set of primers, wherein the first set of primers are fixed or immobilized to a solid support, and wherein the third set comprises at least one production center; and b) contacting the library of nucleic acid analytes with the first set of primers to form more than one first bound entity; c) extending the bound first set of primers by means of template sequences provided by the nucleic acid analytes to form first copies of the analytes; d) contacting the extended first copies with the second set of primers to form more than one second bound entity;
e) extending the bound second set of primers by means of template sequences provided by the extended first copies to form an extended second set of primers; f) separating the extended second set of primers obtained in step e1; g) contacting the extended second set of primers with the third set of primers to form more than one third bound entity; h) extending the third bound entity by means of template sequences provided by the extended second set of primers to form more than one complex comprising the extended third bound entity and the extended set of primers; i) synthesizing from a production center in the second set of primers in the complexes one or more nucleic acid copies under isothermal or isostatic conditions;
j) hybridizing the nucleic acid copies formed in step i) to the array of nucleic acids provided in step a) (i); and k) detecting or quantifying any of the hybridized copies Enz-60 Elazar Rabbani et al., I-"~ng Date: Herewith Page 84 (New Patent Application;) obtained in step j). See this disclosure for a discussion of any of the above-recited elements.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers; b) contacting the nucleic acid _ analytes with the first set of primers to form a first bound entity; c) extending the bound set of first set of primers by means of template sequences provided by the nucleic acid analytes to form first nucleic acid copies of the analytes; d) separating the first nucleic acid copies from the analytes; e) repeating steps b), c) and d) until a desirable amount of first nucleic acid copies have been synthesized; f) hybridizing the nucleic nucleic acid copies formed in step e) to the array of nucleic acids provided in step (i); and g) detecting or quantifying any of the hybridized first nucleic acid copies obtained in step f). These elements are described elsewhere in this disclosure.
Another process for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to sequences of the nucleic acids of interest; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acid analytes, such polymerizing means comprising a first set of primers and a second set of primers;
(iv) means for addition of sequences to the 3' end of nucleic acids; b) contacting the nucleic acid analytes with the first set of primer to form a first bound entity; c) extending the bound set of first set of primers by means of template sequences Enz-60 Elazar Rabbani et al., r ,~~ng Date: Herewith Page 85 (New Patent Application) provided by the nucleic acid analytes to form first nucleic acid copies of the analytes; d) extending the first nucleic copies by the addition of non-template derived sequences to the 3' end of the first nucleic acid copies; e) contacting the extended first nucleic acid copies with the second set of primers to form a second bound entity; f) extending the bound set of second set of primers by means of template sequences provided by the extended first nucleic acid copies to form second nucleic acid copies; g) separating the second nucleic acid copies from the extended first nucleic acid copies; h) repeating steps e), f) and g) until a desirable amount of second nucleic acid copies have been synthesized; i) hybridizing the _ second nucleic acid copies formed in step h) to the array of nucleic acids provided in step (i); and j) detecting or quantifying any of the hybridized second nucleic acid copies obtained in step i). Descriptions for any of the above-recited elements are provided elsewhere in this disclosure.
An illustrative example of this aspect of the present invention would be to bind a poly T primer to poly A mRNA and extend it by Tth DNA polynmerase under conditions that allow it to be used as a Reverse Transcriptase. Thermal denaturation followed by binding of an unextended poly T primer would allow synthesis of another copy by Tth DNA Polymerase. The amount of amplification would be proportional to a) the number of primer binding sites on an individual template molecule b) the efficiency of binding/extension and c) the number of cycles carried out. Thus, with a single primer binding site in a target analyte, 50%
efficiency and 100 cycles denaturation/repriming, the method of the present invention can produce 50 1 ~ cNA copies from a single analyte molecule.
In another aspect of the present invention, primers are used to generate a library of nucleic acids with production centers capable of synthesizing multiple nucleic acid copies that comprise sequences that are either identical or complimentary to sequences in the original analytes. In the first step of this particular aspect of the present invention, the entire population or a subset of the Enz-60 Elazar Rabbani et al., t ..,ng Date: Herewith Page 86 lNew Patent Application population of nucleic acids analytes is used to synthesize 182 strand nucleic acid copies as described previously for linear amplification. In the next step of this aspect of the present invention, the 18t cNA strand is made available for further binding/extension events by the removal or destruction of the template strands.
This can be carried out by a variety of physical, chemical and enzymatic means.
Examples of such methods can consist of but not be limited to denaturation, alkali or RNase treatments. Denaturation can be carried out by exposure to high heat or by the other methods described above for multiple cycles of linear amplification, thereby allowing them to participate in later steps. 1n the next step, primers are annealed to the 1 a' cNA strand in order to synthesize the complementary strands, thereby generating double-stranded cNA copies of the original analyte population.
The primers used for 2"d strand synthesis are designed such that their 5' ends comprise sequences capable of acting as production centers. A description of such production centers is disclosed in Rabbani et al., U.S. Patent Application Serial No.
08/574,443, filed on December 15, 1995 (Novel Property Effecting And/Or Property Exhibiting Compositions for Therapeutic and Diagnostic Uses);
abandoned in favor of U.S. Patent Application Serial No. 08/978,632, filed on November 25, 1997), incorporated herein by reference. An example of a production center that would be particularly useful in the present invention would comprise an RNA
promoter segment.
For example, random hexamer primers for 2"d strand synthesis can have the structure:
5'-promoter- N,NZNaNaNsNs-3' In a preferred mode, the promoter is a phage promoter. The sequences specific for their cognate polymerases are sufficiently short that their addition onto an oligounucleotide being used for priming allows synthesis to remain both efficient Enz-60 Elazar Rabbani et al., i ..,ng Date: Herewith Page 87 (New Patent Application;) and inexpensive. At the same time, they are sufficiently long that they are unique compared to the genomic DNA they are being used with. Also, the phage RNA
polymerises that recognize these promoters are usually single protein molecules that have no requirement for other subunits or cofactors. Of special use in this aspect of the present invention are phage promoter sequences that are recognized by the T3, T7 and SP6 RNA polymerises. These enzymes are well characterized and are commercially available from a number of sources.
For efficient functionality, the promoters cited as examples above should be in double-stranded form. This may be carried out in several different ways. A
potential sequence of events for ane such method is graphically depicted in Figure

4. If the polymerise used for extension has strand displacement activity, the primer binding closest to the 3' end of the 1't strand (Primer A in Figure 4) remains bound to the template, but the other extended primers (Primer B and Primer C) are released from the template in single stranded form. Thus, a given individual template molecule may give rise to a plurality of complementary copies by multiple priming/extension events with two groups of products: essentially double-stranded molecules that comprise the 1 $t cNA strands bound to their complements and single-stranded molecules derived from the displaced strands.
Although initially the displaced strands are in single-stranded form, the continued presence of other primers from either 1't or 2"d strand synthesis could allow further binding/extension events that convert the displaced single strands into double-stranded form. Alternatively, there may have been intermediary purification steps taken to separate extended primers from non-extended primers. For example, separation may be useful to minimize or prevent the synthesis of molecules with promoters at each end. Such double-ended constructs may not transcribe efficiently or may produce nucleic acids that hybridize with each other rather than the target elements of the array. Therefore, the same primers that were used to initiate synthesis of the 1 $t cNA strand can be added to the mixture Enz-60 Elazar Rabbani et al., I-~~~ng Date: Herewith Page 88 (New Patent Application) with the displaced 2"d cNA strands as well as whatever reagents may also be necessary to convert the displaced single-stranded DNA molecules into double-stranded products. Alternatively, random primers without promoters may be used for priming the displaced 2"d cNA strands. The synthesis of a complementary copy for the displaced single strands also converts the promoter segment in the

5'end of these molecules into double-stranded form.
On the other hand, the promoter in the extended primer that remains bound to the original 1 s' cNA strand template (Primer A in Figure 4) needs different processes to render it into a functionally efficient form. For instance, the single-- stranded 3' tail of the 1 °' cNA strand could be digested by the 3' to 5' Exonuclease activity of T4 DNA polymerase.. Upon reaching the double stranded portion, the enzyme could then use its polymerase activity to extend the shortened 3' end by using the promoter segment of primer A as a template thereby generating a double-stranded promoter. In another approach, oligonucleotides can be provided that are complementary to the single-stranded promoter sequences (Figure 5a) or the primers used for 2"d strand cNA synthesis can be designed such that they are self-complementary and form stem loop structures that generate double-stranded functional promoters (Figure 5b). Lastly, the 2"d cNA strands bound to the template can be denatured and the same processes described above for converting the displaced 2"d cNA strands can be used to convert them into double-stranded form.
The creation of functional transcriptional units from the original diverse nucleic acid analytes allows amplification by making multiple transcript copies from each cNA template. By inclusion of the RNA promoter sequence in primers that used the 1$' cNA strand as a template, all the resultant transcripts are also complementary to the 1" cNA strand. However, some target arrays that use defined oligonucleotide sequences as target elements have been designed for the purpose of detecting labeled 1$' cDNA copies of mRNA rather than their Enz-60 Elazar Rabbani et al., f ...,gig Date: Herewith Page 89 (New Patent Application) complements. In such a case, the transcription products of the series of reactions described above can be used as templates to synthesize sequences equivalent to labeled 1" cDNA copies by reverse transcription. As described previously, random or selected primers may find use for this purpose. This conversion step may offer other advantages as well since DNA is known to be more stable than RNA and has relatively less secondary structure compared to RNA.
RNA transcripts or cDNA copies of the RNA transcripts created from the processes described above can either be labeled or unlabeled. When the polynucleotides are unlabeled, they can use UDTs for signal generation. As described previously, the original anlytes may have inherent UDT sequences that may serve this function or the analytes may be modified by the incorporation of non-inherent UDT sequences. On the other hand, the synthetic steps that are carried out in the series of reactions above provide the opportunity to incorporate non-inherent UDTs during either 1 St strand or 2"d strand synthesis by primers with appropriate designs. For example, a primer design for 2"d strand synthesis can have the following structure:
5' promoter-UDT-hexamer-3'.
After binding the primer above to a 1 at cNA strand followed by extension, the transcripts could be generated with the structure:
5' UDT-hexamer-RNA sequence-3'.
Although the transcript shown above has a UDT at its 5' end, other designs allow the transcripts to be synthesized with UDTs in their 3' ends. For instance, this can take place by either the sequence of the primer binding site used for the initial 1 g' strand synthesis being capable of acting as a UDT or by incorporation of a UDT
Enz-60 Elazar Rabbani et al., Fuing Date: Herewith Page 90 (New Patent Application) into the primer that is to be used for 1" strand synthesis. As an example of both methods, a transcription unit can be synthesized from poly A RNA by priming of the 1$' cNA strand with an oligonucleotide primer with the structure:
5' UDT OiigoT-3' and priming of the 2"d cDNA strand by an oligonucleotide primer having the structure 5' promoter-hexamer-3'.
The double-stranded product of 1 °' cNA and 2"d cNA strand synthesis reactions would then have the following structure:
5' promoter- hexamer--2"d strand sequence--PolyA--UDT 3' Transcription from this construct would generate RNA molecules that have the following structure:
5' hexamer--2"d strand sequence --PoiyA--UDT 3' The product above can bind a UDE either through the an inherent UDT (the Poly A
sequence) or through the artificially incorporated UDT. In addition, it should be recognized that the incorporation of UDTs for signal generation can be coupled with incorporation of labeled nucleotides if desired. Thereby, either by direct labeling or by the presence of UDTs, this aspect of the present invention provides for the synthesis of a library of detectable products that will reflect the initial levels of the various nucleic acid analytes of a library.
Enz-60 Elazar Rabbani et al., E ~~~ng Date: Herewith Page 91 (New Patent Application) The use of amplification utilizing RNA synthesis has been previously described by Kwoh and Gingeras, (1989, Proc. Nat. Acad. Sci. USA 86; 1173-1 177; incorporated herein by reference) but the purpose of that work was in diametric opposition to the present invention. In Kwoh and Gingeras, primers with specific sequences were used to synthesize the 2"d cDNA strand in order to amplify a single defined discrete sequence that was of interest. Thus there is no suggestion or recognition of potential benefits of amplification of a diverse population of various nucleic acids.
In a patent application that was filed in the same year as the publication by _ Kwoh and Gingeras, a method was described by van fielder et al. (U.S. Patent No.
5,716,785; incorporated herein by reference) for linear amplification of a general population of RNA targets by including a phage promoter into the primer used for the 1 St cDNA strand. Synthesis of the 2"d strand were carried out either by nicking of the RNA template by RNase H or by hairpin formation at the end of the 1$' cDNA
strands to provide self-priming events. Furthermore, the claims for this patent and a related patent by the same inventors (U.S. Patent No. 5,891,636;
incorporated herein by reference) specifically includes the phrase "without using an exogenous primer". Thus, in these patents there is firstly a requirement of inclusion of a promoter sequence into the primers used for 15' strand synthesis. Secondly there is no appreciation for the use of primers being added to catalyze the 2"d strand synthesis. In fact, there is even a teaching away from this latter concept. In addition, all of the foregoing methods synthesize incomplete copies of the primary analytes as the completeness of the copies made by RNase H are dependent upon the distance of the nick that is closest to the 5' end of the mRNA, only a minority will have representation of the sequences closest to the 5' end of the mRNA.
In addition, there would never be representation of the end itself since it would be used for retaining the RNA fragment/primer closest to the 5' end. Synthesis by means of hairpin formation also has intrinsically incomplete representation of the 5' Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 92 (New Patent Application) end sequences since nuclease degradation of these sequences takes place during elimination of the hairpin. Also, there may be other losses since even nucleases that are considered to be single strand specific are more accurately characterized as having a preference for single-strands since it is well known that there is also some level of activity with segments that are in double-stranded form.
The present invention is in contrast to previously cited art that did not use primers for 2"d strand synthesis. These methods of previous art depended upon the presence of RNaseH to create a second strand or else required self-priming events by a foldback mechanism and subsequent treatment with S1 nuclease or its -equivalent. In the absence of such a nuclease treatment, transcripts made from hairpin derived constructs would be self-complementary and thus incapable of appreciable hybridization to arrays. In contrast to this prior art, the present invention discloses various methods where exogenous primers are used to synthesize the 2"d strand. Also, in some aspects of the present invention, the methods used to synthesize the 2"d strand include means that selectively retain information from the 5' ends of analytes. In addition, the present invention describes the potential for the synthesis of multiple transcription units from a single 1 't strand cNA template thereby providing an additional level of amplification.
It is another aspect of the present invention that the 1 st cNA strands can be actively prevented from creating 2"d cNA strands through a fold-back mechanism by blocking the extendibility of a 1 °t cNA strand. One method of carrying this out is by the addition of a dideoxynucleotide to the 3' terminus of alst cNA copy by terminal transferase. Although this method would prevent a 1$' cNA strand from participating in self-priming reactions, a blocked 1 $t can strand would retain its capability of being used as a template. In this aspect of the present invention, either the primer used for 1" strand cNA synthesis or 2"d strand cNA synthesis can comprise an RNA promoter or other replication center.
Another aspect of the present invention discloses the addition or Enz-60 Elazar Rabbani et al., I-~~wg Date: Herewith Page 93 (New Patent Application) incorporation of artificial primer binding sites to carry out the novel processes described above. For instance, the translation of mRNA into a cDNA copy also frequently includes the terminal addition of a few non-template directed nucleotides into the 3'end of the 1" cNA strand by Reverse Transcriptase. In previous art, these added bases have been used as primer binding sites for cloning of full length cDNA molecules. The addition of a few Cytosine nucleotides at the end of a molecule has been sufficient for the binding and extension of a primer that has 3 Guanosine nucleotides at it 3' end (user Manual for SMART cDNA Technology, Clontech Laoboratories, Inc., Palo Alto, CA ). In this system, aborted or stalled cDNA sequences that were incomplete copies of the original mRNA molecules would not be substrates for the addition reaction by Reverse Transcriptase.
This provided for a more complete representation of the 5' sequences of the original mRNA in a library of cDNA clones.
The non-template derived addition of Cytosine nucleotides to the 1 °' cDNA
strand has been previously used in the process of making a transcription library (Wang et al. 2000, Nature Biotechnology 18; 457-459; incorporated herein by reference). However, this system was basically similar to the method described by van fielder et al., (op. cit.) since a phage promoter was included in the primers used for synthesis of the 1" cDNA strand. As such, this arrangement has the limitation that it has lost the selectivity for molecules that have copied completely their mRNA templates. Primers that bind to interior poly C sequence and initiate extensions are as competent as bindings to poly C's at the end of cDNA (Matz et al., 1999) to synthesize 2"° cDNA strands, thereby creating functional double stranded phage promoters.
In contrast to van fielder et al., and Wang et al., this particular aspect of the present invention provides a promoter in the primer used for the 2"d strand synthesis. Thus, the novel processes that have been disclosed previously can be carried out by the use of a primer for 2"d strand synthesis that comprises oligo dG
Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 94' (New Patent Application) sequences at their 3' end for binding to the termini of 1't cNA strands. In this aspect of the present invention, priming events that derive from the terminal bindings and extensions will lead to double stranded promoters in molecules.
As illustrated in Step (D) in Figure 6, a primer with a T7 promoter can bind to the terminus of the 1" cNA strand. Extension of this primer can create a double stranded molecule where the 3' end of the primer is extended using the cDNA as a template and the 3' end of the cNA is extended using the primer sequences as a' template. The net product of such extensions would be a double stranded transcription unit. On the other hand, Step (E) of figure 6 shows the binding of a primer with a T7 promoter to an internal segment of the cNA with. In this case, although there can be extension from the 3' end of the primer to create a partially double-stranded molecule, the 3' end of the cNA is unable to use the primer as a template, thus leaving the promoter in a non-functional single-stranded form.
One advantage of the system described above is that the non-template addition of nucleotides can be carried out by enzymes that are already present in the reaction mixture. On the other hand, if desired, Terminal Transferase can be added to increase control over the reaction and improve efficiency. When poly A, T or U sequences are already present in either RNA, DNA or cNA copies, it is preferred that the Terminal transferase use dGTP or dCTP. Primers for 2"~
strand synthesis can then be designed whose sequences comprise a promoter and a 3' segment complementary to the sequences added by the Terminal Transferase addition step. The steps of this process are shown in Figure 7, where subsequent extensions to create a double stranded promoter can be carried out as previously described for Figure 6. Also, since the directed addition of nucleotides takes place only where there is either a double stranded end or a free 3' end, only cDNA
molecules that have been completely extended to the ends of the analyte templates will be suitable substrates for terminal addition.
Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 95 (New Patent Application) Since these additions can be longer than those derived from non-template additions by Reverse Transcriptase, the primers used for 2"d strand synthesis can have longer corresponding homopolymeric segments thereby allowing higher temperatures for binding and extension. This heightened stringency should decrease the frequency of priming events with internal sequences in the 1" cNA
template strand and provide higher representation of sequences from the 5' end of the original analytes. Therefore, when terminal transferase is used to generate a primer binding site for 2"d strand synthesis, the promoter can be in either the 1 't strand or the 2~° strand. The step of terminal transferase addition to the 1" cNA
can be carried out while it is still bound to its template as described above, or it can be carried out after destruction of the template or separation of the template from the 1 't cNA strand. This method should continue to enjoy 2"d strand synthesis that is preferentially initiated by primers binding and being extended from the 3' termini of 1't cNA strands. As described previously, UDTs, as well as labeled or unlabeled nucleotides can all be utilized in carrying out this aspect of the present invention. Also, it is contemplated that higher yields of end products can be achieved by repetitions of one or more steps of the various process that are disclosed herein.
Other means that preferentially carry out priming events at the 3' ends of 1 s' strand cNA's may also find use in the present invention. For instance, a cDNA
copy that is a complete copy of its RNA template is a substrate for blunt end ligation by T4 DNA ligase with a double-stranded oligonucleotide. The sequence of the oligonucleotide ligated to the 3' end of the 1" cNA strand can be chosen by the user and can function as a primer binding site for making a 2~° eNA
strand.
Similarly a 3' single-stranded tail in the 1" cNA strand is a substrate for.ligation of a single-stranded DNA oligonucleotide by T4 RNA ligase (Edwards et al., 1991 Nucleic Acids Research 19; 5227-5232; incorporated herein by reference).
Lastly, a double-stranded oligonucleotide with a 3' single-stranded tail can be joined to a Enz-60 Elazar Rabbani et al., f ~,.ng Date: Herewith Page 96 (New Patent Application) 1 St strand cNA through "sticky end" ligation by T4 DNA ligase when the 1 S' cNA
and oligonucleotide tails are complementary. As described previously, these cNA
tails can be derived from non-template additions by Reverse Transcriptase or by Terminal transferase. Illustrative examples of these processes are given in Figure 8. Since all of these processes are dependent upon preferential binding of primers to the 3' ends 1't strand can molecules, the promoter can be in either the 1"
or 2"a cDNA strand.
In another embodiment of the present invention, a 1 S' strand cNA strand is fragmented by physical, chemical or enzymatic means. Examples of enzymatic means can include but not .be limited to restriction enzymes such as Hha I, Hin P1 I
and Mnl I, DNases such as DNase I and nucleases such as S1 nuclease and Mung Bean Nuclease. These fragments can be used as templates for synthesis of a 2"d strand by any of the methods described.previously. For example, hybridization and extension of random primers with T7 promoters can be used with the cNA strand fragments as templates in processes similar to those shown in Figures 4 and 5.
Or if preferred, the homopolymeric addition or ligation steps described above can be carried out to provide specific primer binding sites. Figure 8 is an illustration of this process using the homopolymeric method. Breaking down the 1't strand copy into smaller segments followed by incorporation of a primer during 2"d strand synthesis would provide smaller transcription units. This may be advantageous when using modified nucleotides for signal generation. For instance, when there are long stretches in the template strand that are complementary to the labeled nucleotide, the modification to the nucleotide may cause a blockage in downstream transcription or loss of processivity and result in under-representation of those sequences. In this particular aspect of the present invention, the partition of copies of analyte sequences into smaller individual transcription units allows each of the units to direct RNA synthesis independently thereby creating a more complete representation of the library of various nucleic acid sequences.
Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 97 (New Patent Application) In another embodiment of the present invention, the novel methods disclosed for synthesis of a library are combined with capture methods to provide more efficient synthesis as well as flexibility in changing salts, buffers, enzymes and other components during multistep processes: The present invention discloses the use of a 1$' strand primer that is bound to a solid matrix such as a bead followed by the processes described above. For example, the 3' end of Oligo T sequences bound to a solid matrix can be extended using polyA mRNA as a template. In accordance with the methods of the present invention, this 1 °t cNA
strand is thereupon used as a template for the 2"° cNA strand. When carrying out this aspect of the present invention, a replicative center such as an RNA promoter sequence can be introduced into either the 1 St or 2"d strand depending upon the particular method used. For instance, random primers with promoters in their 5' ends can bind to the extended 1 ~' cDNA strands to create 2"d strands that have a promoter incorporated into them. This process is depicted in Figure 10.
The single-stranded promoter on the 5' ends of the 2"d cDNA strands can be converted into double-stranded form by any of the methods described previously.
For instance, the primer/template complex that remains bound to the bead in Figure can be treated with T4 DNA polymerase, hybridized with an oligonucleotide complementary to the promoter segment or the primer can be designed with self complementary regions. The latter two methods were previously discussed with reference to Figure 5. With regard to the displaced 2"d cDNA strands in Figure 10, the presence of unextended oligo-T tails on the matrix material can provide further binding/extension events since the displaced strands carry poly A sequences on their 3' ends. However, if preferred, more oligo-T can be added whether associated with beads or free in solution. Extension of the oligo-T should ultimately result in conversion of the single-stranded promoters of the displaced 2"d cDNA strands into functional double-stranded forms.
Enz-60 CA 02390141 2002-06-10 __..., ...
Elazar Rabbani et al., Filing Date: Herewith Page 98 (New Patent Application) Another method that can be used in the present invention is to repeat one or more of the steps that have been described in the present invention. For instance, after using a library of analytes to synthesize 1 °' can copies attached to a matrix, the anlytes can be separated from the 1$' cNA copies and used to create another pool of 1 s' cNA copies. Similarly, after synthesis of 2"d can strands, the library of 2"d cNA strands can be separated from the 1 g' can strands fixed to the matrix. All 2"d cNA strands that have copied the 5' ends of the 19' cNA strands will have regenerated the sites that were initially used to bind to the primers linked to the beads. If desired, the 2"d strands can be rebound to the same beads. Since there are likely to be an enormous number of poly T primers on the beads compared to the number of templates used for 1" cNA synthesis, the majority of primers on the matrix remain unextended and can be used for new priming events. Thus, complete copying of these rebound 2"d can strands should allow generation of double-strand promoters at the ends of these molecules without a necessity for the use of T4 to do "trimming". If desired the 1$' cNA strands that are attached to the matrix can be used to generate another pool of 2"° cNA strands. The pool or pools of 2"° can strands can then be added to fresh beads with primers complementary to their 3 ' ends. Again, the extension of the primers attached to the matrix will convert all of the 2~d can strands into double-stranded form including the promoter sequences that were at their 5' ends. Lastly, after a transcription reaction is carried out, the reaction products can be removed and the nucleic acid on the matrix can be used for more transcription reactions thereby accumulating more transcription products.
Although the example above describes priming of an analyte with a poly A
segment by an oligo T primer attached to a matrix, thee primers can also be prepared with one or more discrete bases at their 3' ends. As described previously, these primers can be used as a group that represents all the possible variations or they can be used individually .depending upon whether general Enz-60 Elazar Rabbani et al., Fmng Date: Herewith Page 99 (New Patent Application) amplification or separation into subclasses was desired. The poly A sequence used above is understood to only be an illustrative example. As described previously, the sequences in analytes used for binding of 18' strand primers can be derived from inherent sequences or they may be noninherent sequences in analytes that have been artificially introduced by any of the means that have been described previously. This particular embodiment of the present invention can utilize any of these primer binding sites by appropriate design of the primer sequence bound to the matrix.
In the present invention, the primer sequences for 1 °' strand synthesis can be either directly or indirectly attached to a matrix. Methods for direct attachment of oligonucleotides to matrixes are well known in the art. In addition, beads with covalently attached extendable poly T segments are commercially available from a number of sources. Methods for indirect attachment are also well known in the art. For instance Figure 11 depicts a sandwich method where a primer has two segments, one of which is complementary to a capture segment attached to the matrix and the other is complementary to the poly A segment of the target RNA.
The two segments of the primer may form a continuous nucleotide sequence or there may be a disjunction between the two segments. Hybridization of the two segments of the primer and the complementary sequences on the matrix and the binding site of the analyte can take place simultaneously or they can be carried out in a step-wise fashion. For instance, hybridization of target RNA to the capture element can be carried out in solution followed by capure to the matrix. It is preferred that the segment that is bound to the matrix be rendered incapable of extension. One way this blockage can be carried out is by the use of the 3' end as the attachment point to the matrix as depicted in Figure 11. Binding and extension events can take place as described previously for Figure 10 to synthesize 1 S' and 2"° cDNA copies of the original poly A mRNA. Conversion of the promoter sequences into double-stranded form can also take place as described above.
Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 100 (New Patent Application) Transcription can take place either while the transcription units .are attached to the matrix or if desired separation from the matrix can take place in a step subsequent to the transcription.
Incorporation of an RNA promoter during 18' strand synthesis results in transcripts that comprise sequences that are complementary to sequences in the original analytes. Incorporation of an RNA promoter into the 2"° strand synthesis results in the production of transcripts that comprise sequences that are identical to sequences in the original analytes. As described previously, these can easily be converted into complementary cDNA copies if desired.
It is a further subject of the present invention that transcription units can be synthesized without incorporating a promoter sequence into either the 1" cNA
(as described by Eberwine etal., op. cit.) or the 2"° cNA strand (as described in previous embodiments of the present invention. As shown in step D of Figure 12, when using extended 1 St cNA strands as templates for synthesis of the 2"° cNA
strands, a duplicate of the original primer binding sequence is synthesized.
Thus, in Figure 12 a.polyA segment is created at the 5' ends for both displaced 2"° cNA
strands and for 2~° cNA strands that remain bound to the beads. After removing these 2"° cNA strands, oligonucleotide primers comprising an RNA
promoter and oligo-T sequences can be hybridized to the 2"° cNA strands. The primers may be attached to a matrix or they may be free in solution. Provision of DNA
Polymerase, nucleotides and appropriate cofactors can allow extension of both the 3' ends of the promoter/primers as well as the 3' ends of the cDNA copies thereby creating functional transcriptional units as shown in step F of Figure 12.
Transcription from these DNA molecules will result in products that comprise sequences that are complementary to sequences in the original analytes In previous art the most common use of oligo-T that is attached to a matrix such as cellulose or beads has been for the purpose of a selective isolation of polyA mRNA followed by a release step prior to synthesis of a library. In one Enz-60 CA 02390141 2002-06-10 ' Elazar Rabbani et al., Filing Date: Herewith Page 101 (New Patent Application) instance, a special oligo T primer joined to a T7 promoter was extended using RNA
as template to create a library (Eberwine op.cit.). However, this system put the promoter in close proximity to the capture bead, potentially decreasing its ability to be converted into double-stranded form and/or for it to function as a promoter.
Also, synthesis of the 2"d strand by random priming does not prevent hairpin self-priming. In the absence of a nuclease step, transcription units would direct synthesis of self-complementary RNAs from hairpin template sequences that would be incapable of hybridizing to target arrays. use of the templates for this non-productive synthesis may cause an inefficiency in the amount of effective labeled transcripts A particular benefit of the use of promoters in primers used for 2"° cNA
synthesi present invention is that although 1" cNA strands can be synthesized under conditions that have the potential for self-priming events i.e. creating 2"d cDNA strands by a fold-back mechanism, the absence of a promoter in 1" cDNA;
strand would prevent these constructs from being transcriptionally active.
Thus, only 2"d cDNA strands that are derived from priming events by oligonucleotides with promoter sequences are functional for transcription. This in contrast to the system previously described by Eberwine (op, cit.). Contrariwise, methods have also been described in the present invention that allow the use of a promoter in the 1't strand by either preventing extension of a 1't cNA strand or by facilitating 2"d strand synthesis from priming events at the ends of 1 ~ strand templates.
It is another object of the present invention to provide a method for comparative analysis that requires only a single RNA population to be labeled.
This particular aspect takes advantage of competitive binding by an unlabeled population of RNA. Synthesis of this material can take place by any of the means described in the foregoing work. The particular sequences can be homologous to sequences that are present on the arrays or they may be homologous to sequences that are present in the labeled material. By comparison of hybridization of the labeled Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 102 (New Patent Application) material in the presence or absence of competitor, relative levels of increased or decreased mRNA synthesis can be established relative to the competitor, ie.
differential competition. Adjustments can be made in the relative amounts of unlabeled material being used or the housekeeping genes that are present as controls can allow for normalization values. This method provides the advantage that multiple sequential or parallel hybridizations can be carried out and compared with a single common labeled cantrol population of RNA.
The various steps of the present invention can be carried out sequentially by adding various reagents and incubation steps as required. On the other hand, the series of steps can be segregated by introducing additional steps that either remove or inactivate components of the reaction or where a desired product is separated from a reaction mixture. An example of the former can be heat inactivation of Reverse Transcriptase. An example of the latter can be isolation of RNA/DNA
hybrids by selective matrices. These additional steps can be carried out to either improve the efficiency of subsequent steps or for the purpose of preventing undesirable side reactions.
Although the previous examples have disclosed the utility of a phage promoter in carrying out various aspects of the present invention, a production center is able to operate by other means as well. For instance, various means of introducing UDTs that serve as primer binding sites have been previously described in the context of synthesis of 2"° copy strands followed by RNA
transcription.
These primer binding sites can in themselves serve as production centers for multiple copies of various nucleic acids under isothermal conditions.
For instance the use of primers that are designed to create target-dependent stem-loop structures has previously been disclosed in Rabbani et al., U.S.
Patent Application Serial No. 09/104,067, filed on June 24, 1998 (Novel Processes for Amplifying Nucleic Acid, Post-Termination Labeling Process for Nucleic Acid Sequencing and Producing Nucleic Acid Having Decreased Thermodynamic Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 103 (New Patent Application) Stability; for specific isothermal amplification of selected sequences. The content of the aforementioned Serial No. 09/104,067 is hereby incorporated by reference.
In the present invention, UDTs can be added to the various nucleic acids of a library to carry out the amplification disclosed in Rabbani et al., U.S.
Patent Application Serial No. 09/104,067, cited supra and incorporated herein by reference. Figure 13 is a depiction of a series of reactions that could be used to carry this out. For instance, a UDT can be ligated to a library of poly A mRNA
where the UDT comprises two segments (termed X and Y in this Figure). In the next step, a primer (Primer 1 ) that comprises two segments, a poly T sequence at the 3' end and a segment termed Z at the 5' end is hybridized to the poly A
sequences at the 3' end of.the mRNA and extended by reverse transcription to make a 1 St cNA copy (Steps C and D of Figure 13) that contains the sequnces X' and Y' at the 3' end. Removal of the original template makes the X' segment at the 3' end of the 1 °t cNA copy available for hybridization. A second primer (Primer 2) that has two segments, segment X at the 3' end and segment Y' at the 5 ' end can be annealed and extended to make a 2"° copy (Steps D and E) of Figure 12.
The presence of Primer 2 should also allow a further extension of the 1 't cNA
copy such that a double stranded segment is formed where the Y and Y' segments are capable of self-hybridizing and thereby creating a stem-loop structure with the X
and X' segments in the loop partions as described in Rabbani et al., U.S.
Patent Application Serial No. 09/104,067, cited supra and incorporated herein by reference. Creation of a stem loop at the other end can be carried out by annealing a third primer (Primer 3) which comprises two segments, segment Z at the 3' end and a Poly A segment at the 5' end using a 2"° cNA copy as a template.
The availablity of 2"° cNA copies as templates can be derived from multiple priming events by Primer 2 at the other end (as described in Rabbani et al., U.S.
Patent Application Serial No. 09/104,067, cited supra and incorporated herein by reference, or by denaturation of 'the 1$' and 2"° strands from each other. Extension Enz-60 CA 02390141 2002-06-10 , Elazar Rabbani et al., Filing Date: Herewith Page 104 (New Patent Application) of Primer 3 creates a structure that has the Poly T and Poly A segments forming a stem and the Z and Z' segments forming the loops. Further binding and extension reactions under isothermal conditions can proceed as described previously for unique targets. It should be noted that the particular sequences used for X, Y
and Z are arbitrary and can be chosen by the user. For instance, if the Z segment of Primer 1 used in step C of Figure 13 was designed with X and Y sequences at the 5' end, the unit length amplicon would have X' and Y segments at the 3' end of each strand. As such, amplification could be carried out using only Primer 2.
Another example of the use of non-inherent UDTs being used as primer binding sites for isothermal amplification is shown in Figure 14 for use with the Strand Displacement Amplification system described by Walker et al., in U.S.
Patent No. 5,270,184 herein incorporated by reference. In this particular example, Incorporation of segment X takes place by two different methods. In step B of Figure 14, segment X is introduced by ligation to an analyte of the library.
In step C segment X is attached to a poly T primer and becomes incorporated by strand extension. The presence of the X segment at the 5' end of each end of the amplicon unit allows primer binding by a single Strand Displacement primer.
Methods for the designs of primers with appropriate sequences at their 5' ends have been described by Walker et al., (op. cit.). With regard to the particular enzyme being used as part of the SDA system, the presence of a particular restriction site between primer binding sites may limit the ability of some sequences to be amplified in a reaction designed for general amplification of a library. This may be overcome by choosing relatively uncommon sequences or carrying out parallel reaction with different enzymes.
It should be pointed out that in the examples shown in Figures 13 and 14, the presence of primer binding sites at each end allows exponential amplification.
However, these processes can be changed to linear amplification by designing Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 105 (New Patent Application) amplicons that have binding sites for isothermal amplification at only one end of the amplicon.
Incorporation of a primer binding site that can be used for isothermal production of multiple copies can take place by any of the steps described previously that used a promoter in the example. For instance, Figures 13 and show addition of an isothermal binding site directly to an analyte and also show incorporation of an isothermal binding site during synthesis of a first copy.
Figure 15 shows a similar situation, but in this example segment X is incorporated during 1$' cNA synthesis, segment Q is added after first strand synthesis and segment Z is added during 2"d cNA strand synthesis. As described previously, one or more of these segment can comprise primer binding sites for isothermal synthesis. It should also be pointed out that in Figures 13 through 15 both inherent and non-inherent UDTs were used as part of the examples.
In another aspect of the present invention, UDTs are used as primer binding sites for amplification on an array. In-this particular aspect, each locus on an array comprises two sets of primers. 'The first set of a locus comprises Selective Primer Elements (SPE's) that are specific for a particular analyte. The second set of a locus comprises Universal Primer Elements (UPE's) that are identical or complementary to sequences in UDT elements. As described previously, UDTs can be derived from naturally occurring sequences or they may be artificially incorporated. The SPE"s at a locus would be able to bind to the complementary sequences in the nucleic acids of a library, thereby binding discrete species of nucleic acids to that particular locus of the array. The use of appropriate conditions, reagents and enzymes would allow an extension of an SPE using the bound nucleic acid as a template.
As an example of this aspect of the present invention, Figure 16 depicts an array with three different loci termed Locus P, Locus Q and Locus R. At each of the loci, there is a set of SPE's bound to the array that are complementary to a Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 106 INew Patent Application) particular sequence in cDNA copies made from one of three species of poly A
mRNA termed P, Q and R respectively. In addition, each locus of the array in Figure 16 has a set of UPE's that comprises poly T sequences. Synthesis of a cDNA copy of each of the mRNA templates by Poly T priming of their polyA tails creates cDNA P, cDNA Q and cDNA R respectively. Binding of the 1$' cDNA strand of an analyte to an SPE should be selective for each species at a particular locus.
On the other hand, there should be little or no binding of the cDNA copies to the universal Poly T sequences in the UPE's of the array of Figure 16. The addition of enzymes and reagents for extension should generate 2"d cDNA copies of P, Q and R at the LP, LQ and LR sites on the array by extension of SPE's using the bound cDNA as templates. Each of these 2"d cDNA copies would comprise unique sequences complementary to the 1 S' cDNA strand templates. However, in addition to these unique sequences, the 2"d strand copies would include a common poly A
sequence at their 3' ends. At this stage it may be preferable to remove unhybridized analytes as well as templates used for 2"d strand synthesis. This is most easily carried out by heat denaturation followed by washing steps. The product at this stage is ari array that has extended and un-extended SPE's at each locus where the number of extended SPE's should be in proportion to the amount of the original corresponding analytes. The~extended SPE's can now serve as templates when an unextended poly T UPE is in sufficient proximity. The design and placement of pairs of unique primers for solid phase amplification has been previously described in detail in U.S. Patent No. 5,641,658, hereby incorporated by reference. Methods for synthesis of arrays with two different sequences at each locus has also been described by Gentalen and Chee, 1999 (Nucl. Acids Res. 27;
1485-1491 ) incorporated by reference. The same primer design rules may also be applied to the present invention that uses non-unique primers. Extension of a UPE
with a nearby extended SPE as a template creates a new template that can in turn be used as a template for a nearby unextended SPE. This process can proceed Enz-60 CA 02390141 2002-06-10 .. , .. ..
Elazar Rabbani et al., Filing Date: Herewith Page 107 (New Patent Application) through a series of binding and extension steps that alternatively using SPE's and UPE's to accumulate nucleic acids that are derived from target nucleic acids homologous to the sequences in the SPE at each locus. An illustration of these steps is given in Figures 16 through 19.
Methods for the design and synthesis of arrays for solid phase amplification have been described in U.S. Patent No. 5,641,658 and Weslin et al., 2000, (Nature Biotechnology 18; 199-204; both documents incorporated herein by reference) for utilization of totally unique sets of primers. Methods of assaying the extent of synthesis are also described in these references. For example, labeled precursors can be included in the reaction to synthesize a labeled amplification product. Alternatively, normal precursors can be used with signal generation provided by intercalating dyes binding to amplification products.
This invention provides unique compositions and processes for solid phase amplification. Among such compositions is one that comprises an array of solid surfaces comprising discrete areas, wherein at least two of the discrete areas each comprises a first set of nucleic acid primers; and a second set of nucleic acid primers; wherein the nucleotide sequences in the first set of nucleic acid primers are different from the nucleotide sequences in the second set of nucleic acid primers; wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical.
Previous descriptions for any of the above-recited elements have been given elsewhere in this disclosure, and resort may be made to those descriptions in connection with this process.
Enz-60 CA 02390141 2002-06-10 - -°
Elazar Rabbani et al., Filing Date: Herewith Page 108 (New Patent Application) A related composition of this invention is one comprising an array of solid surfaces comprising a plurality of discrete areas; wherein at (east two of the discrete areas each comprises a first set of nucleic acid primers; and a second set of nucleic acid primers; wherein the nucleotide sequences in the first set of nucleic acid primers are different from the nucleotide sequences in the second set of nucleic acid primers; wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ substantially from each other;
and wherein the nucleotide sequences of the second set of nucleic acid-primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical. See this disclosure above and below for a description of any of the elements in this process.
Related to the last-mentioned compositions are processes for producing two or more copies of nucleic acids of interest in a library comprising the steps of a) providing (i) an array of solid surfaces comprising a plurality of discrete areas;
wherein at least two of the discrete areas each comprises: (1 ) a first set of nucleic acid primers; and (2) a second set of nucleic acid primers; wherein the nucleotide sequences in the first set of nucleic acid primers are different from the nucleotide .
sequences in the second set of nucleic acid primers; wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acids of interest; b) contacting a primer of the first set with a complementary sequence in the nucleic acid of interest; c) Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 109 (New Patent Application) extending the primer in the first set using the nucleic acid of interest as a template to generate an extended first primer; d) contacting a primer in the second set with a complementary sequence in the extended first primer; e) extending the primer in the second set using the extended first primer as a template to generate an extended second primer; f) contacting a primer in the first set with a complementary sequence in the extended second primer; g) extending the primer in the first set using the extended second primer as a template to generate an extended first primer; and h) repeating steps d) through g) above one or more times. Elements above are described elsewhere herein.
Another related process useful for detecting or quantifying more than one nucleic acid of interest in a library comprises the steps of a) providing (i) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of such discrete areas each comprises: ( 1 ) a first set of nucleic acid primers; and (2) a second set of nucleic acid primers; wherein the nucleotide sequences in the first set of nucleic acid primers are different from the nucleotide sequences in the second set of nucleic acid primers; wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and inrherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical; (ii) a library of nucleic acid analytes which may contain the nucleic acids of interest; (iii) polymerizing means for synthesizing nucleic acid copies of the nucleic acids of interest; and (iv) non-radioactive signal generating means capable of being attached to or incorporated into nucleic acids; b) contacting a primer of the first set with a complementary sequence in the nucleic acid of interest; c) extending the primer in the first set using the nucleic acid of interest as a template to generate an extended first primer; d) contacting a primer Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 1 10 (New Patent Application) in the second set with a complementary sequence in the extended first primer;
e) extending the primer in the second set using the extended first primer as a template to generate an extended second primer; f) contacting a primer in the first set with a complementary sequence in the extended second primer; g) extending the primer in the first set using the extended second primer as a template to generate an extended first primer; h) repeating steps d) through g) above one or more times; and i) detecting or quantifying by means of the non-radioactive signal generating means attached to or incorporated into any of the extended primers in steps c), e), g), and h). Elements above are described elsewhere herein.
For many uses, the UPE's will be present on the array during hybridization of the analyte to complementary SPE's. However, there may be circumstances where the presence of UPE's in this step may be deleterious. For example, binding of the diverse nucleic acids of a library should preferably take place only through the action of the SPE's on the array. In contrast to the example given above, there may be cases where due either to the nature of the library or the choice of UPE
sequences, hybridization can take place between the library and the UPE's of an array. This event could result in a loss of efficiency in the reaction by binding of target nucleic acids to inappropriate areas of the array. For instance, the SPE's at a particular locus would be unable to use complementary nucleic acid targets as a template if these targets are inappropriately bound to another physical location.through binding of UPE's,. Furthermore, UPE's would be rendered non-functional by being extended and synthesizing nucleic acid copies that lack complementary to the SPE's at that particular locus.
Accordingly, it is a subject of the present invention that UPE's may be either non-functional or absent during the initial hybridization of a library to the SPE's in the array. In one method of carrying this out, advantage is taken of the universal nature of the UPE's. Although each particular species of SPE is relegated to a specific. area of the array, the UPE's are intended to be present in multiple areas of Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 1 1 1 (New Patent Application) the array. As such, an array can be synthesized where each locus comprises a set of SPE's and a set of chemically activated sites that are compatible with reactive groups on UPE's. After the initial hybridization of nucleic acid targets to their appropriate SPE's, the UPE's with appropriate groups can be added universally to the array by a simultaneous attachment to all of the active sites on the array. An example of compatible modifications that could be used in this aspect of the present invention could be arrays that have maleimide groups at each locus and UPE's that have amine groups attached to their 5' ends.
An alternative approach is for synthesize the array with UPE's that have been modified such that they are temporarily unable to function. For example, the UPE's could be synthesized with 3' POa groups thereby blocking any potential extension reactions. After hybridization of nucleic acids to the various SPE's of the array followed by extension of SPE's, the nucleic acids used as templates could be removed from the reaction. After this step, the 3' end of the UPE's could be rendered functional by removal of the 3' P04 groups by treatment with reagents such as bacteriophage polynucleotide kinase or alkaline phosphatase.
Thereafter, successive reactions can take place as described previously.
An alternative approach would be the use of hybridization properties of nucleic acids. For example, the Tm of hybridization between nucleic acids is a function of their length and base composition. Therefore, the SPE's and UPE's can be designed with Tm's that are sufficiently different that salt or temperature conditions can be used that selectively allow hybridization of the nucleic acids in the sample to SPE's. The salt and temperature conditions can be altered later to allow hybridization to the UPE's on the array and carry out the appropriate series of reactions.
Another example would be the use of competitive hybridization. Nucleic acids or their analogues can be added that are homologous to the UPE's. By either pre-hybridization or by including a high excess of such competitors, the UPE's Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 112 (New Patent Application) should all be occupied with the competitor nucleic acids thereby allowing binding of the nucleic acids of the library to SPE's only. Furthermore, the competitors can be synthesized in such a way that even though they are bound to the UPE's they are unable to serve as templates for extension of the UPEs. Examples of means that can be used for this purpose can include but not be limited to peptide nucleic acids and oligonucleotides with multiple abasic sites. After extension of the SPE's, both the templates used for extension of SPE's and the competitor oligonucleotides bound to the UPE's can be removed concurrently rendering both the extended SPE's and UPE's available for binding to each other.
The poly A RNA in the example shown in Figures 16-19 made use of an inherent UDE in eucaryotic mRNA. As described previously, UDEs can also be added artificially either by polymerization or ligation. For instance, a selected arbitrary sequence can be added to the 5' ends of a library of RNA analytes by the action of T4 RNA ligase. An array could then be used that has SPE's for unique RNA sequences and UPE's with the same sequences as the ligated segment. After localization of the various species of RNA to their appropriate location on an array, an enzyme appropriate for reverse transcription can be added as well as the appropriate buffers and reagents to extend the SPE's thereby synthesizing 1"
strand cDNA copies linked to the array. Removal of the RNA template would then allow the complement of the UPE in the cDNA copy to bind to a nearby UPE on the array followed by a set of reactions as described previously. Since the choice of sequences for artificially added UPE's is of arbitrary nature, this aspect of the present invention can be applied to a simultaneous assay of different pools of analytes by adding different discrete UPE sequences to each library. In contrast to this. the prior art cited above makes no provision for distinguishing between collection of analytes from different sources that have the same sequences. An illustration of an array that could be used for this purpose is given in Figure 20 where two libraries are being compared. One library has been prepared by joining Enz-60 Elazar Rabbani et al., Filing Dates Herewith Page 113 (New Patent Application) sequences for UPE 1 to the nucleic acids and a second library has been prepared that has sequences for UPE 2 joined to the nucleic acids. It should be noted that in Figure 20, Locus 1 of the array has the same SPE's as Locus 9 but they differ in the identity of the UPE where UPE 1 is at Locus 1 and UPE 2 is at locus 9.
This is also true for Locus 2 compared to Locus 10 and so on. Thus, binding of the same sequence can take place at either Locus 1 or Locus 9, but the extent of amplification that will take place at each locus will be dependent upon the amount of bound material that contains the appropriate UPE sequence.
In addition, although the examples above have used RNA or cDNA copies as libraries for this aspect of the present invention, it has been previously disclosed that DNA may also be the initial analyte. As an example of this aspect of the present invention, DNA can be digested with a restriction enzyme to create a library of fragments. A double-stranded UDE can then be ligated to these fragments by the action of T4 DNA ligase. The ligated products can then be denatured and hybridized to an array of SPE's. For example, to investigate potential SNP's at a site "X" on a target nucleic acid, sets of SPE's can be designed that differ by a single nucleotide at their 3' ends. The subsequent efficiency of extensions would then be dependent on how well the nucleotide at site "X" of the target template matched the 3' base of the SPE. As an internal control, a set of SPE's can be designed that will utilize each strand at site "X"
thereby duplicating the information. This process is illustrated in Figure 21.
In this particular example, it is preferred that binding between the nucleic acid and the UPE on the array be prevented since the ligated fragments will have sequences complementary to the UPE's. Examples of means that can be used to carry this out have been described previously whereby UPE's are absent or non-functional during hybridization of the nuclei; acids to the SPE's. On the other hands, the nucleic acids that are being analyzed can be treated such that sequences that are complementary to UPE's are removed. For instance, after the ligation step Enz-60 .... .. _ ~ 02390141 2002-06-10 .... ., ..,.
Elazar Rabbani et al., Filing Date: Herewith Page 114 (New Patent Application) described above, nucleic acids can be treated with a 3' to 5' double-stranded Exonuclease. This should selectively remove sequences complementary to the UPE's while retaining sequences that are identical to sequences in the UPE's.
Regeneration of the sequence complementary to the UPE should then take place only after extension of an SPE. Also as disclosed above, the use of artificial addition of UPE sequences allows the simultaneous analysis of different pools by a selective choice of different UPE sequences for each pool.
It is a further intent of the present invention that rather than choosing specific sequences derived from prior sequence information, a general array can be made that offers complete representation of all possible sequences. For instance, a library of SPE's that are 4 bases in length with permutations of all 4 variable bases would comprise 4 x 4 x 4 x 4 distinct sequences, i.e. a total of 256 permutations.
With a complexity of all potential octamer oligonucleotides with the four variable bases, there would be a total of 256 x 256 for a total of 65,536 permutations.
In prior art, an array covering all the possible amplification products would require two unique primers for each individual amplification. Thusly, there would be a requirement for a total of 65,536 x 65,536 for a total of 4.3 x 109 permutations for pairs of unique octamer primers on the array. Such high numbers may be too .
expensive or too complex to have practical application. On the other hand, the present invention overcomes this limitation by virtue of the use of UPE's.
Accordingly, only the SPE's need to encompass all the possible octamer sequences which results in a requirement for a total of 65,536 different sequences, a number that is easily within the ability of current technology. The number of different nucleic acid that will be amplified at each locus will depend upon the complexity of the library of nucleic acids applied as templates as well as the coriditions used for carrying out amplification. The degree of complexity of the array can also be altered by increasing or decreasing the number of nucleotides comprising the SPE's. Conversely, it has previously been pointed out that a degree of Enz-60 CA 02390141 2002-06-10 . ...
Elazar Rabbani et al., Filing Data: Herewith Page 115 (New Patent Application) differentiation can be achieved by adding one or more discrete bases to the UPE.
For example, the use of a single variable nucleotide at the end of a polyT UPE
would decrease the complexity of the analytes in a library that could be amplified since on average, only one out of three of the various diverse nucleic acid analytes bound to SPE's would be able to carry out strand extension. On the other hand, the inclusion of all 3 sets of UPE's that each carries one of the 3 potential bases in combination with complete representation of octamer SPE's would increase the complexity of arrays from 65,536 sequences to a total of 1.97 x 106 (3 x 65,536) permutations. By using variable nucleotides in the last two nucleotides at the 3' end of the UPE on an array with SPE octamers, the complexity would be 8.0 x (12 x 65,536) permutations. It also should be understood that the complexity of the array can have an incomplete representation of all potential SPE
sequences.
For instance, octomers that have Tm's that are much higher or tower than the average Tm of a random population may be not be desired to be present. Also, octamers that have self-complementary 3' and 5' ends may exhibit poor binding ability. When more than one species of UPE is being used, this aspect can be carried out with amplification carried out simultaneously with each UPE. More preferably, reactions ace carried out in parallel with a given UPE on an array for each set of reactions.
In another aspect of the present invention, a mixed phase amplification is carried out where SPE's at fixed locations on an array are used for 1 s' strand synthesis. but the primers used for synthesis of 2"d strands are not attached to the matrix of the array. ~n this aspect of the present invention, a pool of primers for 2"° strands in solution can make use of normal nucleic acid kinetics to find 1"
strand templates fixed to distinct loci on an array for 2~d strand priming events.
Figures 22-25 show an example of a series of binding and extension reactions with only the SPE's fixed to an array. In this example, SPE-P1 is a primer fixed to Locus P that is complementary to the ( + ) strand of target P and P2 is a Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 116 (New Patent Application) primer that is free in solution and is complementary to the (-) strand of target P.
SPE-Q1 is a primer fixed to Locus Q that is complementary to the ( + ) strand of target Q and Q2 is a primer that is tree in solution and is complementary to the (-) strand of target Q.
It can be seen in Figures 22-25 that the specificity of the reaction and anchoring of the amplfication to a specific locus can be entirely directed by this 1"
strand copying reaction. As such, the identity of the primers that are free in solution are not important as long as they are, capable of synthesizing nucleic acids that can specifically bind to the SPE's on the array. Thus although, unique specific sequences were used in Figures 22-25 for illustration of 2"d strand primingiextension reactions, in this aspect of the invention where a mixed phase amplification is carried out, the primers for synthesis of 2"d strands could also be a carried out by a mixture of UPE's or they can even comprise a pool of or random primers. This particular aspect of the present invention also finds use with general arrays that represent multitudes of variations of sequences. For instance, an array that is created by in situ synthesis as described by Affymatrix can be synthesized with some or all of the 65,536 permutations of an octamer array and then used in conjunction with UPE's in solution.
Another aspect of the present invention discloses novel methods, compositions and kits for the preparation and use of protein and ligand arrays which serve to increase the exposure of the binding substance on the array and decrease non-specific binding to the matrix itself. In one embodiment, chimeric compositions are disclosed that are comprised of two segments, a nucleic acid portion and a non-nucleic portion. The nucleic acid portion is used to achieve a practical and more accessible method for attaching the non-nucleic acid portion to a solid support. In one method of use, the nucleic acid portion is directly bound to the surface of the array where it serves as a linker between the array surface and the non-nucleic acid portions of the chimeric compositions. In addition, due to the Enz-60 Elazar Rabbani et al., Fnmg Date: Herewith Page 1 17 (New Patent Application) phosphate charges of the nucleic acid, each chimeric composition at a locus should exhibit repulsive forces that should minimize interactions between the chimeric compositions.
Since use is being made of its physical properties rather than its sequence identity, any particular sequence can be used generically for all the various chimeric compositions. Information on the identity of the non-nucleic acid portion is not derived from the nucleic acid portion but rather form the spatial location on the array where the chimeric composition has been fixed or immobilized. This is in contrast to prior art, which intrinsically required a diversity of specific sequences for the nucleic acid portion and a subsequent "decoding" of the nucleic acid portion. In another embodiment of the present invention, the nucleic acid portion of the chimeric composition comprises discrete sequences that allow binding of the chimeric composition to the array through hybridization to complementary sequences that are immobilized on the support.
The nucleic acid portion of a chimeric composition can be comprised of deoxynucleotides, ribonucleotides, modified nucleotides, nucleic acid analogues such as peptide nucleic acids (PNAs), or any combination thereof. The sequence of the nucleic acid portion is of completely arbitrary nature and may be chosen by the user. In one aspect of the present invention, advantage is taken of the intrinsic properties of nucleic acid hybridization for the attachment of the non-nucleic acid portion to the solid surface used for the array. Thus, the present invention allows the high specificity, tight binding and favorable kinetics that are characteristic of nucleic acid interactions to be conveyed to a non-nucleic acid portion that does not enjoy these properties.
The non-nucleic acid portion of the chimeric composition of the present invention can be comprised of peptides, proteins, ligands or any other compounds capable of binding or interacting with a corresponding binding partner.
Peptides and proteins can be comprised of amino acid sequences ranging in length from Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 118 (New Patent Application) small peptides to large proteins. This peptides and proteins can also comprise modified amino acids or analogues of amino acids. The amino acids or analogues can comprise any desirable sequence. For instance, the amino acid sequences can be derived from enzymes, antibodies, antigens, epitopes of antigens, receptors and glycoproteins. When peptides or proteins are used as the non-nucleic acid portion of the chimeric composition, the sequences of the nucleic acid portion are of arbitrary nature and have no correspondence to the amino acid sequences of the peptides or proteins. Other molecules besides peptides and proteins may also find use in the present invention. Examples of other constituents that could be used for the non-nucleic acid portion can comprise but not be Limited to ligands of MW
of 2000 or less, substrates, hormones, drugs and any possible protein binding entity.
As described previously, the particular sequence of the nucleic acid is determined by the user. In one method of use of the present invention, each individual species that is used as the non-nucleic acid portion can be covalently joined to a unique nucleic acid sequence. Hybridization of a the nucleic acid portion of the chimeric composition to a complementary sequence at a particular locus on an array thereby determines the identity of the particular species of the non-nucleic acid portion that is now bound to that locus. For example, one hundred different chimeric compositions can be synthesized that each comprises a unique peptide and a unique nucleic acid sequence. Hybridization can then be carried out with an array that has one hundred different loci, where each locus has nucleic acids complementary to one of the unique nucleic acid sequences.
Hybridization- thereby results in the localization of each unique peptide to one particular locus on the array, transforming a nucleic acid array into a peptide array.
A useful method for selection of sequences that could be used for the nucleic acid portion has been described by Hirschhorn et al., (op.cit.) hereby incorporated by reference. Also, since no relationship is required between the non-nucleic portion and the nucleic acid portion, a different set of one hundred chirneric compositions Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 119 (New Patent Application) can be designed that have different species used for the non-nucleic acid portion but use the same set of one hundred sequences for the nucleic acid portion. In this way, a generic nucleic acid array can be used to create different peptide arrays by changing the identities of the chimeric compositions.
Alternatively, non-nucleic acid protein binding substances can be attached to oligonucleotides which all comprise the same sequence. For example, chimeric compositions with various non-nucleic portions could be synthesized where the nucleic acid portion of each chimeric compositions comprised a common poly T
sequence. The matrix can be prepared so that the oligonucleotides at each site consist of complementary Poly A sequences. The chimeric compositions can then be applied to the matrix using an addressable arraying system that has been described by Heller et al. in U.S. Patent No. 5,605,662 (herein incorporated by reference). By these means, each particular chimeric composition can be applied individually to the matrix using an electronically controlled system and immobilized through hybridization to the appropriate site.
The chimeric compositions at a particular locus of an array do not have to be completely uniform in nature, i.e. an oligonucleotide sequence can be attached to several different species of non-nucleic acid portions. For example, a series of one hundred peptides can be placed on the array in only four different sites by making Pool 1 with twenty-five peptides conjugated to oligonucleotide 1, Pool 2 with twenty-five peptides conjugated to oligonucleotide 2, Pool 3 with twenty-five peptides conjugated to oligonucleotide 3 and Pool 4 with twenty-five peptides conjugated to oligonucleotide 4. Attachment of the various pools of chimeric compositions to each locus can be carried out by having oligounucleotide 1, 2, and 4 comprising unique sequences complementary to different oligonucleotides at each site or as described above, an addressable arraying system can be used to localize each pool using nucleic acid portions with identical sequences. The chimeric compositions comprised of nucleic acid and non-nucleic acid portions can Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 120 (New Patent Application) be synthesized using any method known to those skilled in the art. Methods that may find use with the present invention are described in a review by Tung, C.-H.;(2000 Bioconjugate Chemistry 11, 5, 605-618) and Engelhardt et al., U.S.
Patents 5,241,060, issued August 31, 1993 and Pergolizzi et al., U.S. Patent Application Serial No. 08/479,995, filed June 7, 1995, for Analyte Detection Utilizing Polynucleotide Sequences, Composition, Process and Kit, based on priority U.S. Patent Application Serial No. 06/491,929, filed May 5, 1983, alf incorporated herein by reference. In one approach, peptides and oligonucleotides are synthesized separately using standard automated procedures and then covalently bonded together. For example, a thiol group can be added either to the 5'-terminus of the oligonucleotide or internally in the nucleic acid portion of the chimeric composition. Addition of a maleimido group to the N-terminus or in an internal position of the peptide allows a reaction with the thiol group of the oligonucleotide to form a chimeric composition comprised of a nucleic acid and a peptide (Eritja et al., ( 1991 ) Tetrahedron, 47; 4113-4120. Arar et al.; ( 1993) Tetrahedron Lett 34;
8087-8090, Ede et al., (1994) Bioconjugate Chemistry 5; 373-378, Stetsenko and Gait, (2000) J. Org. Chem. 65; 4900-4908). Alternatively the chirneric composition can be prepared by the stepwise addition of amino acids and nucleotides on the same solid support, (de la Torre et al., (1994) Tetrahedron l-ett 35; 2733-2736,. Bergmann and Bannwarth (1995) Tetrahedron Lett. 36; 1839-1842, Robles et al., (1999) Tetrahedron 55; 13,251-13,264, Antopolsky et al., (1999) Helv. Chim Acta 82; 2130-2140). In these publications each of which is incorporated by reference herein, the peptide was synthesized first followed by the addition of bases to synthesize the oligonucleotide portion. In standard peptide synthesis, the N-terminus and the side chains of the amino acids are protected by Fmoc and tent-butyl groups respectively. At each cycle the Fmoc group is removed with 20% piperidine and the side chains are deprotected with 90%
trifluoroacetic acid. However when both oligonucleotides and peptides were synthesized as part Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 121 (New Patent Application) of a single composition, different chemistries had to be used. For example, base labile Fmoc and 9-fluorenylmethyl groups were used as the amino acid side chain protecting groups to avoid exposing the DNA to strong acids (de la Torre, op cit.;
de la Torre et. al., 1999 Bioconjugate Chem.10; 1005-1012; Robles et al op cit.), all such publications being incorporated by reference herein. Methods for making chimeric compositions of peptides fused to PNA analogues of nucleic acids have been described by Cook et al. in U.S. Patent No. 6,204,326, incorporated herein by reference. Furthermore, chimeric compositions comprised of nucleic acids and peptides can be synthesized directly on a solid surface to create an array using the methods described by Sundberg et al in U.S. Patent No. 5,919,523 incorporated herein by reference.
The solid support can be any material used for arrays including, but not limited to nylon or cellulose membranes, glass, synthetic, plastic, metal. The materials can be opaque, reflective, transparent or translucent. They can be porous or they can be non-porous. Nucleic acids that are either part of chimeric compositions or meant to be complementary to chimeric compositions can be affixed to the solid support by any previously known methods used to prepare DNA
arrays.
Binding of analytes to appropriate binding partners can be carried out in either a mixed phase or a liquid phase format. For instance, the present invention has disclosed the direct fixation of binding substances to the array by the use of rigid arm linkers and chimeric compositions. The binding substance on the array (the solid phase) can be exposed to a solution Ithe liquid phase) that contains the analytes of interest. Interactions between the binding substance on the array and analytes in solution can then later be quantified. Examples of the interactions that may find use in the present invention can comprise but not be limited to peptide-protein, antigen-antibody, ligand-receptor or enzyme-substrates. For example, an array can be prepared with a series of peptides to determine their ability to bind to Enz-60 Elazar Rabbani et al., Firing Date: Herewith Page 122 (New Patent Application) a particular antibody. The array is incubated in a solution containing the antibody followed by washing away the unbound material. Detection of the antibody bound to various components on the array can then be carried out by any of a number of conventional techniques. For instance, in this example the antibody that is applied to the array can be labeled with biotin for indirect detection, or a fluorescent compound for direct detection. Alternatively, the antibody analyte is unlabeled and a secondary antibody can be utilized which either has a fluorescent label for direct detection or indirect label such as biotin. Thus, in this example the antibody-antigen interaction occurs with the antigen bound to the solid matrix.
The present invention has also disclosed the use of chimeric compositions that are indirectly bound to the array through hybridization of the nucleic acid portions of the chimeric compositions to complementary nucleic acids fixed or immobilized to the array. These can be used in the in the same mixed phase format that has been described above by hybridization of the chimeric compositions to the array followed by binding of analytes. However, the use of hybridization to immobilize the chimeric compositions to specific loci on the array allows the use of a completely liquid phase format for binding of analytes to the chimeric compositions. In this way, the chimeric compositions can be combined with the target molecules in solution under optimal conditions for interactions between the analyte and the non-nucleic acid portions of the chimeric compositions. The resultant solution, containing the chimeric compositions free in solution as well as the chimeric compositions that are bound into complexes with the analytes, can then be applied to the matrix and the various chimeric compositions will be localized to various locations on the array through hybridization to the nucleic acid portion to complementary sequences on the array. An illustration of this process is given in Figure 28.
The hybridization can be carried out under mild conditions, which will not interfere with the ligand-receptor or protein-protein complex. Protein-protein Enz-60 CA 02390141 2002-06-10 ....
Elazar Rabbani et al., Filing Date: Herewith Page 123 (New Patent Application) interactions are generally characterized by low Km's, in the order of magnitude of 10'fi to 10'9. In this aspect of the present invention, the protein interactions can occur in solution rather than on a solid surfaces which will allow superior kinetics of binding and will also allow a wider variety of conditions for protein binding than can be obtained in the mixed format. Also, by chimeric compositions and analytes together in solution, direct interaction or interference with the matrix ~s avoided, thereby decreasing the background. Therefore, to use the example cited before, the solution containing the antibody target is combined with. a solution containing the chimeric composition. Thus, by using the methods of the present invention, the proteins will remain in solution throughout the process preventing any problems associated with dehydrating the protein bound to the solid matrix.
The method of the present invention can be used to study many systems that involve interactions between compound. These can include but not be limited to antigen-antibody relationships, protein-protein interactions, enzyme-substrate receptor-ligand interactions, ligand-receptor, hormone-receptor, carbohydrate-lectins, drug screening, and patterns of expression of proteins in a cell or tissue.
Another method of use of the present invention is that instead of using unique nucleic acid portions for each individual non-nucleic acid portion, one specific binding substance can be combined with various nucleic acid sources to form a group of chimeric compositions with a common non-nucleic acid portion and a unique nucleic acid portion. Each particular chimeric composition can be combined with an analyte from a different source and applied to the array by hybridizing the nucleic acid portions to their complementary sequences on the array. The proteins bound to the array can then be detected following standard procedures. By these means, the amount of targets from each source that can interact with the binding substance in the chimeric compositions can be simultaneously determined..
For instance, a set of twenty different compositions can be synthesized where each member of the set will have a different nucleic acid portion but the Enz-60 Elazar Rabbani et al.; Filing Date: Herewith Page 124 (New Patent Application) same peptide. Another set can be made with a different peptide that is linked to twenty other nucleic acid portions. More sets can be made on the same basis.
Protein extracts can then be made from twenty different tissues and each extract can be combined with a different member of the set of chimeric compositions.
Thus, the nucleic acid portion serves as a marker for not only the peptide but also for the particular tissue that was used as the source. For instance, a group of sets can be made with peptides that have affinities for different receptors. After incubation of the mixtures with the chimeric compounds, the mixtures are applied to the array and detected. In this way, each particular receptor that is being studied can be quantified and compared simultaneously between various tissues.
Alternatively, the same nucleic acid sequence can be used in common for each source by using the addressable system described previously, and carrying out hybridization to each locus after addition of each individual reaction mixture.
The same method can be applied to tissues or cell cultures that are from the same source but are treated differently. For example, in a drug discovery .program, nine different drugs can be added to individual cell cultures to determine the effect on specific proteins. Chimeric compositions are designed and synthesized with peptides that are known to react with each of proteins that is to be monitored. As _ in the previous example, a specific nucleic acid sequence will serve as a marker for each peptide and each particular treatment. The proteins are extracted from each of the ten cell cultures (nine drug treated plus an untreated control) and incubated with the chimeric compositions. The mixtures are applied to the array and the amount of analyte bound to the corresponding peptides at each locus of the array is measured for the various drug conditions. If desired, the present invention can also be used for the isolation of analytes. This can be carried out by either disrupting the interaction between the analyte and the non-nucleic acid portion of the chimeric compositions or by denaturing the nucleic acid portion from the complementary sequence fixed or immobilized to the array. It is also contemplated Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 125 (New Patent Application) that removal of chimeric compositions from the array may also allow the reuse of the array in other experiments.
In further detail, this invention provides novel chimeric compositions and processes using such chimeric compositions. One such composition of matter comprises an array of solid surfaces comprising a plurality of discrete areas, wherein at least two of such discrete areas comprise: a chimeric composition comprising a nucleic acid portion; and a non-nucleic acid portion, wherein the nucleic acid portion of a first discrete area has the same sequence as the nucleic acid portion of a second discrete area, and wherein the non-nucleic acid portion has a binding affinity for analytes of interest.
Another composition of matter comprises an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of the discrete areas comprise a chimeric composition hybridized to complementary sequences of nucleic acids fixed or immobilized to the discrete areas, wherein the chimeric composition comprises a nucleic acid portion, and a non-nucleic acid portion, the nucleic acid portion comprising at least one sequence, wherein the non-nucleic acid portion has a binding affinity for anaiytes of interest, and wherein when the non-nucleic acid portion is a peptide or protein, the nucleic acid portion does not comprises sequences which are either identical or complementary to sequences that code for such peptide or protein.
Mention should be made of a process for detecting or quantifying analytes of interest, the process comprising the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas, wherein at least two of such discrete areas comprise a chimeric composition comprising a nucleic acid portion, and a non-nucleic acid portion; wherein the nucleic acid portion of a first discrete area has the same sequence as the nucleic acid portion of a second discrete area;
and wherein the non-nucleic acid portion has a binding affinity for analytes of interest; b) a sample containing or suspected of containing one or more of the Enz-60 CA 02390141 2002-06-10 ... . .. ..
Elazar Rabbani et al., Filing Date: Herewith Page 126 (New Patent Application) analytes of interest; and c) signal generating means; 2) contacting the array a) with the sample b) under conditions permissive of binding the analytes to the non-nucleic acid portion; 3) contacting the bound analytes with the signal generating means; and 4) detecting or quantifying the presence of the analytes.
Another process for detecting or quantifying analytes of interest comprises the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of such discrete areas comprise a chimeric composition comprising a nucleic acid portion; and a non-nucleic acid portion;
wherein the nucleic acid portion of a first discrete area has the same sequence as the nucleic acid portion of a second discrete area; and wherein the non-nucleic acid portion has a binding affinity for analytes of interest; b) a sample containing or suspected of containing one or more of the analytes of interest; and c) signal generating means; 2) labeling the analytes of interest with the signal generating means; 3) contacting the array a) with the labeled analytes under conditions permissive of binding the labeled analyzes to the non-nucleic acid portion;
and 4) detecting or quantifying the presence of the analytes.
Another process for detecting or quantifying analytes of interest comprises the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of such discrete areas comprise nucleic acids fixed or immobilized to such discrete areas, b) chimeric compositions comprising: i) a nucleic acid portion; and ii) a non-nucleic acid portion; the nucleic acid portion comprising at least one sequence, wherein the non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when the non-nucleic acid portion is a peptide or protein, the nucleic acid portion does not comprise sequences which are either identical or complementary to sequences that code for the peptide or protein; c) a sample containing or suspected of containing the analytes of interest; and d) signal generating means; 2) contacting the array with the chimeric compositions to hybridize the nucleic acid portions of the chimeric Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 127 (New Patent Application) compositions to complementary nucleic acids fixed or immobilized to the array;
3) contacting the array a) with the sample b) under conditions permissive of binding the analytes to the non-nucleic acid portion; 4) contacting the bound analytes with the signal generating means; and 5) detecting or quantifying the presence of the analytes.
Another process for detecting or quantifying analytes of interest comprises the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of the discrete areas comprise nucleic acids fixed or immobilized to the discrete areas, b) chimeric compositions comprising i) a nucleic acid portion; and ii) a non-nucleic acid portion, the nucleic acid portion comprising at least one sequence, wherein the non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when the non-nucleic acid portion is a peptide or protein, the nucleic acid portion does not comprise sequences which are either identical or complementary to sequences that code for the peptide or protein; c) a sample containing or suspected of containing the analytes of interest; and d) signal generating means; 2) contacting the chimeric compositions with the sample b) under conditions permissive of binding the analytes to the non-nucleic acid portion; 3) contacting the array with the chimeric compositions to hybridize the nucleic acid portions of the chimeric compositions to complementary nucleic acids fixed or immobilized to the array; 4) contacting the bound analytes with the signal generating means; and 5) detecting or quantifying the presence of the analytes.
Another useful process comprises the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of the discrete areas comprise nucleic acids fixed or immobilized to the discrete areas, b) chimeric compositions comprising i) a nucleic acid portion; and ii) a non-nucleic acid portion; the nucleic acid portion comprising at least one sequence, wherein the non-nucleic acid portion has a binding affinity for analytes of interest, and wherein Enz-60 CA 02390141 2002-06-10 . ... _ . . .
Elazar Rabbani et al., Filing Date: Herewith Page 128 INew Patent Application) when the non-nucleic acid portion is a peptide or protein, the nucleic acid portion does not comprise sequences which are either identical or complementary to sequences that code for the peptide or protein; c) a sample containing or suspected of containing the analytes of interest; and d) signal generating means; 2) contacting the array with the chimeric compositions to hybridize the nucleic acid portions of the chimeric compositions to complementary nucleic acids fixed or immobilized to the array; 3) labeling the analyzes of interest with the signal generating means; 4) contacting the array with the labeled analytes to bind the analytes to the non-nucleic acid portion; and 5) detecting or quantifying the presence of the analytes.
Another process for detecting or quantifying analytes of interest comprises the steps of 1 ) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of the discrete areas comprise nucleic acids fixed or immobilized to the discrete areas, b) chimeric compositions comprising: i) a nucleic acid portion; and ii) a non-nucleic acid portion; the nucleic acid portion comprising at least one sequence, wherein the non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when the non-nucleic acid portion is a peptide or protein; such nucleic acid portion does not comprise sequences vvhich are either identical or complementary to sequences that code for the peptide or protein; c) a sample containing or suspected of containing the analytes of interest; and d) signal generating means; 2) contacting the array with the chimeric compositions to hybridize the nucleic acid portions of the chimeric compositions to complementary nucleic acids fixed or immobilized to the array;
3) labeling the analytes of interest with the signal generating means; 4) contacting the array with the labeled analytes to bind the analytes to the non-nucleic acid portion;
and 5) detecting or quantifying the presence of the analytes.
The elements recited in the last several chimeric compositions and processes using such chimeric compositions are described elsewhere in this disclosure.
Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 129 (New Patent Application) The examples which follow are set' forth to illustrate various aspects of the present invention but are not intended in any way to limit its scope as more particularly set forth and defined in the claims that follow thereafter.
Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 130 (New Patent Application) DESCRIPTION OF THE PREFERRED EMBODIMENTS
Example 1 Amplification of a library of RNA targets with 2"d strand synthesis carried out by random primers with T7 promoter sequences 1 ) First strand synthesis Two mixtures of 250 ng of rabbit globulin mRNA (Life Technologies, Rockvifle, MD) and 200 ng of Oligo (dT)za (In house or purchased?) in 5 u1 were heated at 70°C for minutes followed by a 2 minute incubation on ice. This material was then used in 10 u1 reactions containing 50 mM Tris-HCI (pH 8.3), 75 mM KCI, 3 mM MgClz, 10 mM DTT, 600 uM dNTPs and 120 units of Superscript II RNase H' Reverse Transcriptase (Life Technologies, Rockville, MD) with incubation at 42°C for 60 minutes.
2) Second strand synthesis KOH was added to the reactions for a final concentratiion of 200 mM.
Incubation was carried out at 37°C for 30 minutes followed by neutralization with an equimolar amount of glacial acetic acid. Primers with the following sequence were used for 2"d strand synthesis:
5'-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGN,.s-3' Primers with the sequence above (TPR primers) consist of a T7 promoter sequence at their 5' ends and 9 nucleotides with random sequences at their 3' ends. 400 pmoles of TPR primers and other appropriate reagents were added for a final reaction mix of 30 u1 containing 86.6 mM Tris-HCI (pH 7.6), 32 mM KCI, 200 mM
KOAc (??), 15.6 mM MgClz, 3.3 mM DTT, 10 mM Dithioerythritol (DTE), 10 mM
(NHa)zSOo, 0.15 mM -NAD, 200 ug/ml nuclease-free BSA (Bayer, Kankakee, IL), Annealing was carried out by heating the mixture to 65°C and slow cooling to Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 131 (New Patent Application) room temperature followed by incubation on ice for 5 minutes. Extension of the primers was carried out by addition of 1.2 u1 of 10 mM dNTPs, 4 units of E.
coli DNA ligase (New England Biolabs, Beverly, MA) and either 12 units of DNA
polymerase I (New England Biolabs, Beverly, MA) or 6 units of the Exo (-) version of the Klenow fragment of DNA Polymerase I (New England Biolabs, Beverly, MA).
Incubation was carried out at 15°C for 5 minutes followed by 37°C for 120 minutes. The reactions were puriifed by extraction with PhenoI/Chloroform with Phase-Lock Gels(Eppendorf, Westbury, NY) and Ethanol precipitated.
3) Transcription Transcription was carried out by using the BioArray High Yield Transcription Kit (T7) (ENZO Diagnostics, Farmingdale, NY) following the manufacturers instructions with a final volume of 40 u1. The reaction mixes also contained 10 uCi of 3H-ATP
with a specific activity of 45 Ci/mMol (Amersham Pharmacia, Piscataway, NJ).
Incorporation was measured by addition of 5 u1 of the transcription reaction to 1 ml of 10% TCA, 50ug/ml Poly A, 5mM EDTA followed by incubation on ice for 30 minutes. Precipitates were collected on 25 mm glass fiber filters (Whatman, Lifton, NJ) followed by three washes with 5% TCA and three washes with ethanol 4) Results and conclusions Sample 1 with DNA polymerase I 4,243 cpm Sample 2 with Exo (-) Klenow 19,662 cpm This example demonstrated that RNA transcripts were obtained from a library of nucleic acids by the steps described above and that under the conditions used, the Exo (-) version of Klenow resulted in more product compared to the use of DNA
polymerase I.
Enz-60 Elazar Rabbani et al., 1-umg Date: Herewith Page 132 (New Patent Application) Example 2 Amplification of a library of RNA targets with 1'~ strand synthesis using Oligo-T magnetic beads and 2"d strand synthesis carried out by random primers with T7 promoter sequences 1) Preparation of Beads 50 u1 of Dynal Oligo (dT)ZS magnetic beads (Dynal Inc., Lake Success, NY) were washed two times with 100 u1 of Binding Buffer (20mM Tris-HCI (pH 7.51, 1.0 M
LiCI, 2mM EDTA) and then resuspended in 50 u1 of Binding Buffer.
2) Binding of RNA to Beads .
RNA targets were prepared by diluting I ug of mouse poly A RNA (Sigma Chemical Co, St. Louis, MO) or I ug of wheat germ tRNA (Sigma Chemical Co, St. Louis, MO) into RNase-free Hz0 (Ambion, Austin, TX) for a final volume of 50 u1, and heating the RNA solution at 65°C for 5 minutes. The RNA solution was combined with the beads prepared in Step 1 and mixed for 15 minutes at room temperature with a Dynal Sample Mixer (Dynal Inc., Lake Success, NY). Unbound material was removed by magnetic separation with a Dynal Magnetic Particle Concentrator (Dynal, Inc. Lake Success, NY) followed by two washes with 200 u1 of Wash Buffer B (10 mM Tris-HCI (pH 7.5), 150 mM LiCI, 1 mM EDTA) and three washes with 250 u1 of First Strand Buffer (50rnM Tris-HCl (pH 8.3), 75mM KCI, 3mM
MgClz) 3) First stand Synthesis The beads from Step 2 were resuspended in 50mM Tris-HCI (pH 8.31, 75mM KCI, 3mM MgClz. 10 mM DTT, 500 uM dNTPs and 400 units of Super Script II RNase H' Reverse Transcriptase (Life Technologies, Rockville, MD) and incubated for 90 minutes at 42°C.
Enz-60 Elazar Rabbani et al., Filing Date. Herewith Page 133 (New Patent Application) 4) Second Strand Synthesis RNA templates were removed by heating the First Strand Synthesis reaction mixture of step 3 at 90°C for 5 minutes followed by removal of the supernatant after magnetic separation. The beads were washed two times with 100 u1 of Buffer C (70mM Tris-HCI (ph 6.9) 90 mM KCI, 14.6 mM MgCIz,10 rnM DTE, 10 mM (NHa)zS04 and 200 ug/ml nuclease-free BSA) and resuspended in 50 u1 of Random Priming Mix A (86.7 rnM Tris-HCI (pH 7.6), 113.3 mM KCI, 17 mM
MgClz,11.3 mM DTT, 11.3 mM (NH4)2S0~, 227 ug/ml nuclease-free BSA) containing 360 pmoles of TPR primers. Primers were allowed to anneal on ice for 15 minutes. Unbound primers were removed by magnetic separation. The beads were resuspended in 50 u1 of Random Priming Mix A (without the TPR primers) with 10 units of the Klenow fragment of DNA Polymerase I (New England Biolabs, Beverly, MA) and 400 mM dNTP's. Incubation was carried out for 5 minutes at 4°C, 30 minutes at 15°C, and 30 minutes at 37°C. For some samples, an additional 25 u1 of Oiigo T magnetic beads prepared as described in Step 1 were washed with Buffer C and added to the reaction mix. Also, for some samples, 3 units of T4 DNA Polymerase (New England Biolabs, Beverly, MA) and 2 u1 of a lOrnM stock of dNTPs were added to the reaction mixtures. Samples with these further steps were incubated for 30 minutes at 37 °C. At the conclusion of the varied reactions, the beads were magnetically separated from the reagents.and the beads were used to carry out transcription assays.
5) Transcription Synthesis Transcription reactions were carried out by resuspending the beads in reagents from the BioArray High Yield Transcription Kit (T7) (ENZO Diagnostics, Farmingdale, NY) using the manufacturer's instructions with a final volume of u1. The reaction mixtures also contained 10 uCi of 3H-ATP with a specific activity Enz-60 .. ' ~ 02390141 2002-06-10 . .,. ...., ..
Elazar Rabbani et al., Filing Date: Herewith Page 134 INew Patent Application) of 45 Ci/mMol (Amersham Pharmacia, Piscataway, NJ). Extent of transcription was measured by using TCA precipitation as described previously.

6) Results Sample Target Extra T4 DNA cprn Incorporated Beads polymerase 1 Poly A (-) (-) 8,535 2 Poly A (-) ( + ) 15,483 3 Poly A 1 + ) (-) 16,048 4 Poly A ( + ) 1 + ) 18,875 tRNA ( + ) ( + ) 2,548

7) Conclusions This example demonstrated that transcripts were obtained from a library of nucleic acids by the steps described above. Addition of extra beads can increase the amount of synthesis. The reaction can be carried out without a T4 DNA
polymerization step but the amount of synthesis can be increased by the addition of such a reagent.
Example 3 Dependency on Reverse Transcriptase for Amplification of a library of RNA targets with Oligo-T magnetic beads and random primers with T7 promoter sequences 11 Preparation of Beads This step was carried out as described in Step 1 of Example 2, except the amount of beads was increased to 100 u1 for each reaction Enz-60 Elazar Rabbani et al., I-uing Date: Herewith Page 135 (New Patent Application) 2) Binding of RNA to beads RNA targets were prepared by diluting I ug of mouse poly A mRNA (Sigma Chemical Co, St. Louis, MO) into nuclease-free Ha0 (Ambion lnc., Auistin TX) for a final volume of 50 u1 , and heating the RNA solution at 65°C for 15 minutes. The RNA solution was combined with the beads prepared in Step 1 and mixed for 15 minutes at Room Temperature with a Dynal Sample Mixer (Dynal Inc., Lake Success, NY). Unbound material was removed by magnetic separation.followed by two washes with 200 u1 of Wash Buffer B and two washes with 100 u( of First Strand Buffer.
3) First strand synthesis This step was carried out as described in step 3 of Example 2 except that a pair of duplicate samples had the Reverse Transcriptase omitted 4) Second strand synthesis RNA templates were removed by heating the First Strand Synthesis reaction mixture of step 3 at 90°C for 4 minutes followed by removal of the supernatant after magnetic separation. The beads were washed two times with 100 u1 of Wash Buffer B and resuspended in 50 u1 of Random Priming Mix A containing 360 pmoles of TPR primers. Primers were allowed to anneal on ice for 15 minutes.
Unbound primers were removed by magnetic separation and the beads were washed twice with 100 u1 of cold Buffer D (20mM Tris-HCI (pH 6.9), 90 mM KCI, 4.6 mM MgCla" 10 mM (NH4)aS04. The beads were then suspended in 40 u1 of Buffer C that also contained 1 mM dNTPs and 10 units of the Klenow fragment of DNA Polymerase I (New England Biolabs, Beverly, MA). Incubation was carried out for 5 minutes at 4°C, 30 minutes at 15°C, and 30 minutes at 37°C. The reaction was carried out further by the addition of 2 u1 (6 units) of T4 DNA Polymerase Enz-60 Elazar Rabbani et al., 1-icing Date: Herewith Page 136 (New Patent Application) (New England Biolabs, Beverly, MA) and 2 u1 of a 10 mM stack of dNTPs, followed by incubation for 30 minutes at 37°C.
5) Transcription Synthesis The beads were washed two times with 100 u1 of Wash Buffer B and once with 100 u1 of 10 mM Tris-HCI (pH 7.5). The beads were resuspended in 10 u1 of 10 mM Tris-HCI (pH 7.5) and mixed with reagents from a BioArray High Yield Transcription Kit (T7) (ENZO Diagnostics, Farmingdale, NY) using the manufacturer's instructions. The volume of the reaction was 30 u1 and the incubation was carried out for 2 hours at 37°C.
6) Resuits and Conclusions Analysis of the reaction was carried out .by gel electrophoresis of 10 u1 of the transcription reaction using 196 Agarose in 0.5x TBE buffer. The results of this experiment are in Figure 27 for duplicate samples and demonstrate that transcripts were obtained from a library of nucleic acids by the steps described above and this synthesis was dependent upon the presence of Reverse Transcriptase activity.
Example 4 Multiple rounds of synthesis of 2"d strands by random primers with promoters Steps 1, 2 and 3 for Preparation of beads, binding of mRNA and 1 °t strand synthesis were carried out as described in steps 1 through 3 of Example 3.
4) Second strand synthesis After 1 °' strand synthesis, the liquid phase was removed by magnetic separation and the beads resuspended in 100 u! of Detergent Wash No.1 (10 mM Tris-HCI (pH
7.5), 1 % SDS) and heated at 90°C for 5 minutes.. The supernatant was removed by magnetic separation and the beads were washed with 100 u1 of Detergent Enz-60 Elazar Rabbani et al., , .~mg Date: Herewith Page 137 (New Patent Application) Wash No.2 (40 mM Tris-HCI (pH 8.0), 200 mM KCI, 4.2 mM EDTA, 0.01 Tween 20, 0.01 % Nonidet P40). The beads were washed two times with 100 u1 of Wash Buffer B and resuspended in 50 u1 of Random Priming Mix A containing 360 pmoles of TPR primers. Primers were allowed to anneal on ice for 15 minutes.
Unbound primers were removed .by magnetic separation and the beads were washed twice with 100 u1 of cold Buffer D (20mM Tris-HCI (pH 6.9), 90 mM KCI, 4.6 mM MgCl2,10 mM DTT, 10 rnM (NHa)zS04). The beads were then suspended in 40 u1 of Buffer C that also contained 1 mM dNTPs and 10 units of the Klenow fragment of DNA Polymerase I (New England Biolabs, Beverly, MA). Incubation was carried out for 5 minutes at 4°C, 30 minutes at 15°C, and 30 minutes at 37°C.
The reaction was carried out further by the addition of 2 u1 (6 units) of T4 DNA
Polymerase (New England Biolabs, Beverly, MA) and 2 u1 of a 10 mM stock of dNTPs, followed by incubation for 30 minutes at 37°C. The beads were then washed two times with 100 u1 of Wash Buffer B, resuspended in 50 u1 of 10 mM
Tris-HCI (pH 7.5) and heated at 90°C for 5 minutes. The supernatant was removed after magnetic separation and store as supernatant No.1. The beads were then washed once with 100 u1 of Detergent Wash No.2, two times with 100 u1 of Wash Buffer B and resuspended in 50 u1 of Random Priming Mix A containing 3fi0 pmoles of TPR primers. Primer annealing and extension was carried out as described above. The beads were then washed two times with 100 u1 of Wash Buffer B, resuspended in 50 u1 of 10 mM Tris-HCI (pH 7.5) and heated at 90°C for minutes. The supernatant was removed after magnetic separation and store as supernatant No.2. The series of washes, annealing and extension steps were carried out again using the steps described above. The beads were then washed two times with 100 u1 of Wash Buffer B, resuspended in 50 u1 of 10 mM Tris-HCI
(pH 7.5) and heated at for 5 minutes. The supernatant was removed after magnetic separation and stored as supernatant No.3.
Enz-60 Elazar Rabbani et al., racing Date: Herewith Page 138 (New Patent Application) 5) Synthesis of complements to the 2"d strands A pool was created by combining supernatant No.1, supernatant No.2 and supernatant No.3. This pool comprises a library of 2"d strands free in solution with T7 promoters at their 5 ' ends and poly A segments at their 3' ends. Fresh magnetic beads with poly T tails were prepared and annealed to the pool of 2"d strands by the same processes described in Steps 1 and 2 of Example 2.
Extension was then carried out by resuspension of beads in 50 u1 of Buffer C
that also contained 1 mM dNTPs and 10 units of the Klenow fragment of DNA
Polymerase I (New England Biolabs, Beverly, MA). Incubation was carried out at 37°C for 90 minutes. Transcription was then carried out as described in step 5 of Example 3 except the reaction volume was reduced to 20 u1.
6) Results and Conclusions The results of this experiment are in Figure 28 and demonstrated that transcripts were obtained from a library of polyA mRNA by the steps described above. This example demonstrated that a library of 2"d strands was obtained after multiple rounds of 2"° strand synthesis, isolated free in solution and then used to create functionally active production centers Example 5 Additional RNA synthesis from transcription constructs The library of transcription constructs described in Example 4 were used for a second round of transcription. After removal of transcription products for analysis in Example 4, the beads were resuspended in 100 u1 of lOrnM Tris-HCI (pH 7.5) and left overnight at 4°C. The next day, the beads were washed with 100 u1 of Detergent Wash No.2, resuspended in 100 u1 of Detergent Wash No.1 and heated at 42°C for 5 minutes followed by two washes with 100 u1 of Detergent Buffer No.2, two washes with 100 u1 of Wash Buffer B and two washes with 100 u1 of Enz-60 Elazar Rabbani et al., , .,mg Date: Herewith Page 139 (New Patent Application) mM Tris-HCI (pH 7.5). A transcription reaction was set up as described previously with a 20 u1 volume.
Results and Conclusions Results of the transcription reaction are shown in Figure 29 and show that the nucleic acids synthesized in Example 4 were stable and could be used for additional transcription synthesis.
Example 6 Terminal Transferase addition of poly G tail to 1 n strands for binding of primers with T7 promoter 1 ) Preparation of beads 150 u1 of Dynal Oligo (dTlzs magnetic beads (Dynal Inc., Lake Success, NY) were washed two times with 150 u1 of Binding Buffer and resuspended in 75 u1 of Binding Buffer.
2) Binding of RNA to beads RNA targets were prepared by diluting 3 u1 of 0.5 ug/ul mouse poly A RNA
(Sigma Chemical Co, St. Louis, MO) with 32 u1 of RNase-free H20 (Ambion, Austin, TX) and 40 u1 of Binding Buffer, and heating the RNA solution at 65°C for 5 minutes.
The RNA solution was combined with the beads prepared in Step 1 and mixed for 30 minutes at room temperature.
31 First strand synthesis Unbound material was removed by magnetic separation.followed by two washes with 200 u1 of Wash Buffer B and one wash with 100 u1 of First Strand Buffer.
The beads were resuspended in a 50 u1 mixture of 50 mM Tris-HCI (pH 7.51, 75 mM KCI, 3mM MgCl2, 10 mM DTT, 500 uM dNTPs and 400 units of Super Script II
RNase H' Reverse Transcriptase (Life Technologies, Rockville, MD) and incubated Enz-60 Elazar Rabbani et al., riling Date: Herewith Page 140 (New Patent Application) for 90 minutes at 42°C. At the end of the 1 °t strand synthesis reaction, the liquid phase was removed by magnetic separation and the beads resuspended in 100 u1 of Detergent Wash No.1 and heated at 90°C for 5 minutes. The supernatant was removed by magnetic separation and the beads were washed with 100 u1 of Detergent Wash No.2, two times with 100 u1 of Wash Buffer B and resuspended in 300 u1 of 10 mM Tris-HCI (pH 7.5).
4) Second strand synthesis Two methods were used for carrying out second strand synthesis. The first method was as described for the previous examples, I.e the use of TPR primers that have a T7 promoter on their 5' ends and random sequences at their 3' ends.
The second method was the use of T7-C9 primers that have a T7 promoter at their 5' ends and a poly C segment at their 3' ends. The sequence of the T7-C9 primers is as follows:
5'GGCCAGTGAATTGTAATACGACTCACTATAGGGATCCCCCCCCC-3' The product of Step 3 was divided into two portions. The first portion (Sample No.1 ) consisted of 100 u1 and was set aside to be used for random priming.
The second portion (the remaining 200 u1) was processed further by magnetically separating the buffer from the beads and resuspending the beads in 100 u1 and adding 100 u1 of Poly A Mix (1.6 ug/ul Poly A, 10 mM Tris-HCL (pH 7.5), 0.5 M
LiCI, 1 mM EDTA). The Poly A was obtained from (Amersham Pharmacia, Piscataway, NJ) and had an average length of 350 nucleotides. The beads and Poly A were mixed together for 30 minutes at room temperature with a Dynal Sample Mixer (Dynal Inc., Lake Success, NY). The beads were washed two times with Wash Buffer B and resuspended in 200 u1 of 10m Tris-HCI (pH 7.5 ). This was divided into two 100 u1 portions, Sample No.2 and Sample No.3. Sample Enz-60 Elazar Rabbani et al., . ,~mg Date: Herewith Page 141 (New Patent Application) No.3 was processed further by magnetically separating the buffer from the beads and resuspending the beads in an 80 u1 reaction mixture using reagents and directions from the 3' Oligonucleotide Tailing System (6NZ0 Biochem, Farmingdaie, NY 11561 ) with 0.5mM dGTP present. Sample No.3 was incubated for one hour at 37°C followed by removal of the reagents by magnetic separation. The beads were then resuspended in 100 u1 of Detergent Buffer No.1 and heated at 90°C for 3 minutes. The beads were then washed once with 100 u1 of Detergent Wash No.2 and twice with 100 u1 of Wash Buffer B. Sample No. 3 was resuspended in 100 u1 of 10 mM Tris-HCI (pH 7.5). All three samples (Sample No.1, Sample No.2 and Sample No.3) were washed once with 100 u1 Wash Buffer E (100 mM Tris-HCI
pH7.4) 20 mM KCI, 10 mM MgClz, 300 mM (NH4)ZSOo) and then resuspended in 50 u1 of Buffer E. Primers for 2"d strand synthesis were added to each sample:
4u1 of 100 pMole/ul of TPR primers to Sample No.1 and 4u1 of 10 pMole/ul of T7-C9 primers to Samples No.2 and No.3. Samples were then incubated on ice for 15 minutes followed by one wash with 100 u1 of ice cold Buffer E and one wash with ice cold Buffer D. Each sample was resuspended in 40 u1 of Buffer D that also contained 1 mM dNTPs and 200 units of the Klenow fragment of DNA Polymerase I (New England Biolabs, Beverly, MA). Incubations were carried out for 30 minutes at 15°C followed by 30 minutes at 37°C.
All three samples were further processed by the addition of 2 u1 (3 units) of T4 DNA polymerase (Source, Location) and 2 u1 of 10 mM dNTPs followed by incubation at 37°C for 30 more minutes. Samples were washed twice with 100 u1 of 10 mM Tris-HCI (pH 7.5). A Transcription reaction was set up as described previously with a 20 u1 volume.
5) Results and Conclusions Analysis of the reaction was carried out by gel electrophoresis with 2u1 and 10u1 samples of the transcription reaction using 1 % Agarose in 0.5x TBE buffer.
The Enz-60 Elazar Rabbani et al., t-wing Date: Herewith Page 142 (New Patent Application results of this experiment are in Figure 30 and demonstrated that non-inherent UDTs were added to the ends of a library of 1" strand copies by the methods described above. The non-inherrent UDTs served as primer binding sites for primers with poly C at their 3' ends for synthesis of a library of 2"d strands . The difference in the amount of RNA transcription between Samples No.2 and No. 3 serves as a further indication that comparatively little priming took place at internal sites under the conditions used.
Example 7 Terminal Transferase addition of poly G tai! to 1" strands for binding of primers with T7 promoter (Incorporation assay) The transcription products of Example 6 were analyzed by gel electrophoresis as shown in Figure 30. To obtain numerical evaluation of the method described in that example, the libraries attached to the beads in Samples No.1, No.2 and No.3 were used in another transcription reaction using 3H-incorporation. Transcription was carried out as described in Example 3.
The results were as follows:
Random priming Sample No.1) 6,660 cpm T7-C9 primers without TdT addition step Sample No.2 1,144 cpm T7-C9 primers with TdT addition step Sample No.3 21,248 cpm This second assay agrees with the conclusions of Example 6; i.e. the T7-C9 primers can be used in the present method and more priming took place with the terminally added poly G sequences compared to internal sequences.
Enz-60 Elazar Rabbani et al., r ~nng Date: Herewith Page 143 (New Patent Application) Example 8 Incorporation of promoters after 2"d strand synthesis 1 ) Preparation of beads Preparation of beads for each sample was carried out as described in step 1 of Example 3 2) Binding of RNA to beads In each sample, 1 ug of poly mRNA was bound to beads as described in step 2 of Example 3 with the addition of having 120 units of Prime RNase Inhibitor (Eppendorf, Westbury, NY) present.
3) First strand synthesis First strand synthesis was carried out as described in step 3 of Example 3 except the reaction was also supplemented with 120 units of Prime RNase Inhibitor 4) Second strand synthesis Poly dG addition was carried out as described for sample No. 3 in Example 6.
Second strand synthesis was performed as described in Example 6 except that 80 pMoles of primers were used in 100 u1 reactions. For Samples No. 1 and No.2, the 2nd strand primers were the T7-C9 primers previously described. For Samples No.3 and No.4, the 2nd strand primers were C9 primers with the sequence:
5'-CCCCCCCCC-3'. At the end of the reaction, all samples were washed twice with 100 u1 10 mM Tris-HCI (pH 7.5).
5) Third strand synthesis Samples No.2, No.3 and No.4 were processed further by resuspension of the beads in 26 u1 of 10 mM Tris-HCI (pH 7.5) and heating at 90°C for 3 minutes.
The second strands released by this process were isolated apart from the beads by Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 144 (New Patent Application) magnetic separation and mixed with 40 pMoles of 3'° strand primers for a final volume of 30 u1. For Sample No.3, the 3'° strand primers were T7-Tzs primers with the sequence 5' GGCCAGTGAATTGTAATACGACTCACTATAGGGATC(T)zs-3' For Samples No.2 and No.4, the 3'° strand primers were T3-Tzs primers with the sequence:
5' CTCAACGCCACCTAATTACCCTCACTAAAGGGAGAT(T)zs-3' After mixing, Samples No.2, No.3 and No.4 were kept on ice for 15 minutes.
Extension reactions were then set up in 1 x M-MuLV Buffer (New England Biolabs, Beverly MA) with 10 units of M-MuILV Reverse Transcriptase (New England Biolabs, Beverly MA) and 1 mM of each dNTP in a final volume of 40 u1.
Incubation was carried out for one hour at 37°C. 6 units of T4 DNA
Polymerase (New England Biolabs, BeverIy,MA) were added to Samples No.1, No.2, No.3 and No.4 and incubation carried out for a further 15 minutes at 37°C.
Reactions were stopped by the addition of EDTA (pH 8.0) to a final concentration of lOmM. The DNA from Samples 2, No.3 and No.4 was then purified by adjusting the volumes to 150 u1 by adding appropriate amounts of 10mM Tris-HCI. Reactions were mixed with an equal volume of Phenol:chloroform:isoamyl alcohol (25:24:1 ) and transferred to 2 ml Phase Lock Gel Heavy tubes(Eppendorf, Westbury, NY). Tubes were vorteed for 1-2 minutes and centrifuged for 10 minutes at 16,000 rpm in a microfuge. The aquaenus phase was then transferred to another tube and DNA
precipitated with Ethanol and Ammonium Acetate.
Enz-60 Elazar Rabbani et al., ruing Date: Herewith Page 145 (New Patent Application) 6) Transcription Beads (Sample No.1 ) and precipitates (Samples No.2, No.3 and No.4) were resuspended with components from the BioArray High Yield Transcription Kit (T7) (ENZO Diagnostics, NY) and transcription carried out in a 20 u1 volume following the manufacturer's directions with the addition of 5 uCi 3H-CTP , 20 Ci/mMol (Amersham Pharmacia Biotech, Piscataway, NJ). In addition some reactions were carried out as described above, but T3 RNA polymerase from the BioArray High Yield Transcription Kit (T3) (ENZO Diagnostics, NY) was substituted. Reactions were carried out for 120 minutes at 37°C
7) Results 2"d strand3'd strand Sample Primer PrimerRNA Polym CPM
No.

No.1 T7-C9 ----- T7 12,392 No.2 T7-C9 T3-Tzs T7 29,160 No.2 T7-C9 T3-TZS T3 14,784 No.3 C9 T7-Tzs T7 22,622 No.4 C9 ' T3-TZS T3 12,221

8) Conclusions This example demonstrated that a promoter can be introduced during 3'd strand synthesis to create functional production centers. This example also demonstrated that in addition to a T7 promoter, a T3 promoter was also functional in the present method. This example also demonstrated that different production centers could be introduced into each end of a construct (Sample No.2) and both production centers were functional.
, Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 146 (New Patent Application) Example 9 Multiple rounds of 2"~ strand synthesis with Thermostable polymerases 1 ) Preparation of Beads, Binding of RNA to Beads and First strand synthesis were carried out as described in Example 8.
2) Second strand synthesis and Recycling Poly dG addition was carried out as described for sample No. 3 in Example 6 and the beads with tailed 3' ends were used for 2"° strand synthesis under various conditions. 50 u1 Reactions mixes were set up as follows: Sample No.1 consisted of 1 x Taq PCR Buffer (Epicentre, Madison, WI), 3m M MgClz, 1 x PCR Enhancer (Epicentre, Madison, WI), 0.4 mM dNTPs, 40 pMoles C9 primers and 5 units of Master AmpT"" Taq DNA Polymerase (Epicentre, Madison, WI); Sample No.2 was the same as sample No.1 except 100 pMoles of C9 primers were used; Sample No.3 consisted of 1 x Tth PCR Buffer (Epicentre, Madison, WI), 3mM MgClz, 1 x PCR Enhancer (Epicentre, Madison, WI), 0.4 mM dNTPs, 40 pMoles C9 primers and units of Master AmpT"" Tth DNA Polymerase (Epicentre, Madison, WI); Sample No.4 was the same as sample No.3 except 100 pMoles of C9 primers were used Samples No.1 and No.3 went through one binding/extension cycle while samples No.2 and No.4 went through 5 such cycles. Each binding extension/extension cycle was carried out in a thermocycler under the following conditions:
2 minutes at 90°C
5 minutes at 4°C
5 minutes at 37°C
5 minutes at 50°C
minutes at 72°C
At the end of each cycle, samples No.2 and No.4 were briefly shaken to resuspend the beads. After the completion of either 1 or 5 cycles, the mixtures were heated at 90°C for 3 minutes and the aqueous portion collected after magnetic separation.
Enz-60 Elazar Rabbani et al., ruing Date: Herewith Page 147 (New Patent Application) Each sample was phenol extracted and ethanol precipitated as described previously in step 5 of Example 8 for samples No.3 and No.4.
3) Third strand synthesis Pellets were resuspended in 26 u1 of 10 mM Tris-HCI (pH 7.51 and T7-Tzs primers were added. For Samples No.1 and No.3, 40 pMoles of T7-Tzs were added; for Samples No.2 and No.4, 400 pMoles of T7-Tzs were added. Third strand synthesis was then carried out by the addition of MuLV, MuLV buffer and dNTPS
as described in step 5 of Example 8.
4) Transcription Transcription was carried out as described previously without the addition of radioactive precursors. Analysis of the reaction from each sample was carried out by gel electophoreis as described previously and shown in Figure 31.
51 Conclusions This example demonstrated that thermostable poiymerases could be used for 2"° strand synthesis in the methods described above. This example also demonstrated that by increasing the amount of primers and the number of cycles the amount of RNA copies derived from the original library of nucleic acids was increased.
Example 10 Levels of transcription derived from sequential rounds of 2"° strand synthesis 1 ) Preparation of Beads, Binding of RNA to Beads and First strand synthesis were carried out as described in Example 8 except the amount of analytes and reagents for each reaction was increased two-fold. Preparation of 1't strands for 2"° strand synthesis was carried out as described previously for sample 3 in Example 6.
Enz-60 Elazar Rabbani et al., Filing Date: Herewith Page 148 (New Patent Application) 2) Second strand synthesis Second strand synthesis was carried out as described for Sample No.3 in Example 8. Separation and isolation of the 2"d strand products was carried out as described in Example 8 and set aside as Sample No.1. Fresh reagents were then added to the beads and another round of 2"d strand synthesis was carried out.
The products of this second reaction were separated from the beads and designated Sample No.2. The beads were then used once more for a third round of synthesis.
The products of this reaction were set aside as Sample No.3.
3) Third strand synthesis Samples No.1, No.2 and No.3 were used as templates for 3'd strand synthesis in individual reactions with the reagents and condition previously described in Example 8. As mentioned above, the starting material in the present example was twice the amount used in example 8 and as such the amounts of all reagents were doubled for this reaction as well. For example, 80 pMoles of T7-T2s primers were used. Purification of the products from each reaction was carried out as described in Exampls 8.
4) Transcription Transcription reactions were carried out as with the BioArray High Yield Transcription Kit (T7) fENZO Diagnostics, NY). The DNA was used in a 20 u1 final reaction volume which was incubated for 2 hours at 37°C. Gel analysis was then used to evaluate the amount of synthesis that was a result of each round of 2"a strand synthesis described above. For purposes of contrast, various amounts of the transcription reaction (4 u1 and 10 u1) were analyzed and in addition equvalent amounts of the DNA template that were not used in transcription reactions were also included. The results of this are shown in Figure32.
Enz-60 CA 02390141 2002-06-10 ..
Elazar Rabbani et al., Filing Date: Herewith Page 149 (New Patent Application) 5) Conclusion This example demonstrated that the 2"d strands made in each round of 2"d strand synthesis were substantially equal in their ability to be used to synthesize a library with functional production centers. Figure 32 also shows the contrast between the amount of transcript and the original DNA templates used for this synthesis thereby demonstrating the high levels of synthesis from each template.
Example 11 use of Reverse Transcriptases from various sources Preparation of Beads, Binding of RNA to Beads and 1 S' strand synthesis were carried out as described in Example 6 except that Reverse Transcriptases from various sources were used for 1 g' strand synthesis reactions. 2"d strand synthesis was carried out as described in Example 6 for sample No.2 , i.e Terminal Transferase addition followed by binding and extension of T7-C9 primers. A
list of the various Reverse Transcriptases and their sources is given below.
1 ) Superscript II [RNaseH(-) MuLV] (Life Technologies, Rockville, MD) 2) RNase H (+) MuLV (Life Technologies, Rockville, MD) 3) RNase H ( + ) MuLV (New England Biolabs, Beverly, MA) 4) Enhanced AMV (Sigma, St. Louis, MO) 5) AMV (Life Technologies, Rockville, MD) 6) AMV (Sigma, St. Louis, MO) 7) Omniscript (Cliagen 8) Display THERMO-RT Display Systems Biotech,

9) Powerscript [RNaseH(-) MuLV) (Clontech laboratories, Each 2"d stand synthesis was carried out in the buffer provided by the manufacturer for each Reverse Transcriptase with the exception of the New England Biolabs version of RNase H ( + ) MuLV which was used in the buffer Enz-60 ~ 02390141 2002-06-10 Elazar Rabbani et al., Filing Date: Herewith Page 150 (New Patent Application) provided for the Life Technologies version of RNase H ( + ) MuLV. Further processing and transcription reactions were as previously described in Example 6.
The results of this experiment re shown in Figure 33.
Conclusions A variety of different Reverse Transcriptases ccould be used ira conjunction with the methods of the present invention.
Many obvious variations will no doubt be suggested to those of ordinary skill in the art in light of the above detailed description and examples of the present invention. All such variations are fully embraced by the scope and spirit of the invention as more particularly defined in the claims that now follow.
Enz-60 SEQUENCE LISTING
<110> Schimmel, Paul Wakasugi, Keisuke <120> Human Aminoacyl-tRNA Synthetase Polypeptides Useful For The Regulation of Angiogenesis <130> 00-221 <140>
<141>
<160> 58 <170> PatentIn Ver. 2.0 <210> 1 <211> 5174 <212> DNA
<213> Artificial Sequence <220>
<221> CDS
<222> (3428)..(5035) <220>
<223> Description of Artificial Sequence: human full-length TyrRS in pET20B
<400> 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa c:ttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta r_gtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctc:gccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctca agcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttact;ca tatatacttt ag<~ttgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt tt~~cgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca cJtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgc:gc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaa catgccc 2990 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg ga<~cagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt cca cagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccatt:ca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360 tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420 tatacat atg ggg gac get ccc agc cct gaa gag aaa ctg cac ctt atc 3469 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu His L~=_u Ile acc cgg aac ctg cag gag gtt ctg ggg gaa gag aag ctg aag gag ata 3517 Thr Arg Asn Leu Gln Glu Val Leu Gly Glu c;lu Lys Leu Lys Glu I1e ctg aag gag cgg gaa ctt aaa att tac tgg gga acg gca acc acg ggc 3565 Leu Lys Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly aaa cca cat gtg get tac ttt gtg ccc atg tca aag att gca gac ttc 3613 Lys Pro His Val Ala Tyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe tta aag gca ggg tgt gag gta aca att ctg ttt gcg gac ctc cac gca 3661 Leu Lys Ala Gly Cys Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala tac ctg gat aac atg aaa gcc cca tgg gaa ctt cta gaa ctc cga gtc 3709 Tyr Leu Asp Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val agt tac tat gag aat gtg atc aaa gca atg ctg gag agc att ggt gtg 3757 Ser Tyr Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val 95 100 105 1'10 ccc ttg gag aag ctc aag ttc atc aaa ggc act gat tac cag ctc agc 3805 Pro Leu Glu Lys Leu Lys Phe I:Le Lys Gly Thr Asp Tyr Gln Leu Ser aaa gag tac aca cta gat gtg tac aga ctc tcc tcc gtg gtc aca cag 3853 Lys Glu Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln cac gat tce aag aag get gga get gag gtg gta aag cag gtg gag cac 3901 His Asp Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His cct ttg etg agt gge ctc tta tac ece gga <a g cag get ttg gat gaa 3949 Pro Leu Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu gag tat tta aaa gta gat gcc caa ttt gga ggc att gat cag aga aag 3997 Glu Tyr Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Ar_g Lys att ttc acc ttt gca gag aag tac ctc cct gca ctt ggc tat tc:a aaa 4045 Ile Phe Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys cgg gtc cat ctg atg aat cct atg gtt cca gga tta aca ggc agc aaa 4093 Arg Val His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys atg agc tct tca gaa gag gag tcc aag att gat ctc ctt gat cgg aag 4141 Met Ser Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys gag gat gtg aag aaa aaa ctg aag aag gcc ttc tgt gag cca gga aat 4189 Glu Asp Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn gtg gag aac aat ggg gtt ctg tcc ttc atc aag cat gtc ctt ttt ccc 4237 Val Glu Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro ctt aag tcc gag ttt gtg atc cta cga gat gag aaa tgg ggt gcfia aac 4285 Leu Lys Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn aaa ace tac aca get tac gtg gac ctg gaa aag gac ttt get gca gag 4333 Lys Thr Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala A_l.a Glu gtt gta cat cct gga gac ctg aag aat tct gtt gaa gtc gca ctg aac 4381 Val Val His Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn aag ttg ctg gat cca atc cgg gaa aag ttt aat acc cct gcc ctg aaa 4429 Lys Leu Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys aaa etg gec agc get gce tac eca gat cec tca aag cag aag cca atg 4477 Lys Leu Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pr_o Met gcc aaa ggc cct gcc aag aat tca gaa cca gag gag gtc atc cca tcc 4525 Ala Lys Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Pr_o Ser cgg ctg gat atc cgt gtg ggg aaa atc atc act gtg gag aag cac cca 4573 Arg Leu Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys H:is Pro gat gca gac agc ctg tat gta gag aag att gac gtg ggg gaa get gaa 4621 Asp Ala Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu A:La Glu cca cgg act gtg gtg agc ggc ctg gta cag ttc gtg ccc aag gag gaa 4669 Pro Arg Thr Val Val Ser Gly Leu Val Gln Phe Val Pro Lys G.Lu Glu ctg cag gac agg ctg gta gtg gtg ctg tgc aac ctg aaa ccc cag aag 4717 Leu Gln Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln Lys atg aga gga gtc gag tcc caa ggc atg ctt ctg tgt get tct ata gaa 4765 Met Arg Gly Val Glu Ser Gln Gly Met Leu Leu Cys Ala Ser I:Le Glu ggg ata aac cgc cag gtt gaa cct ctg gac cct ccg gca ggc tct get 4813 Gly Ile Asn Arg Gln Val Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala cct ggt gag cac gtg ttt gtg aag ggc tat gaa aag ggc caa cca gat 9861 Pro Gly Glu His Val Phe Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp gag gag ctc aag ccc aag aag aaa gtc ttc gag aag ttg cag get gac 4909 Glu Glu Leu Lys Pro Lys Lys Lys Val Phe Glu Lys Leu Gln A.La Asp ttc aaa att tct gag gag tgc atc gca cag tgg aag caa acc a<~c ttc 4957 Phe Lys Ile Ser Glu Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe 495 500 '_~05 510 atg acc aag ctg ggc tcc att tcc tgt aaa tcg ctg aaa ggg ggg aac 5005 Met Thr Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly G:ly Asn att agc ctc gag cac cac cac cac cac cac tgagatccgg ctgctaacaa 5055 Ile Ser Leu Glu His His His His His His agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct 5115 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat 5174 <210> 2 <211> 536 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: human full-length TyrRS in pET20B

<400> 2 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu His Leu Ile Th r Arg Asn Leu Gln Glu Val Leu Gly Glu Glu Lys I~eu Lys Glu Ile Le a Lys Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro His Val Ala Tyr Phe Val Pro Met Ser Lys ile Ala Asp Phe Leu Lys Ala Gly Cys Glu Val Thr I1e Leu Phe Ala Asp Leu His Ala Tyr Leu Asp Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr Tyr Glu Asn Val Ile Lys Ala Met Leu Glu tier Ile Gly Val Pro Leu Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser Lys Glu Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Va1 Val Thr Gln H_is Asp Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His Pro Leu 145 150 1.55 160 Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu G1u Tyr 165 170 1'75 Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Arg Lys I_Le Phe Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Va1 His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys G:Lu Asp Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Leu Lys Ser Glu Phe Val Ile Leu Arg Asp Glu Lys 'rrp Gly Gly Asn Lys Thr Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu V<~l Val His Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu 305 310 ;515 320 Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr I'ro Ala Leu Lys Lys Leu Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met A_La Lys Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Pro Ser Ar_g Leu Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg 385 390 i95 400 Thr Val Val Ser Gly Leu Val Gln Phe Val Pro Lys Glu Glu Leu Gln Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln Lys Met Arg Gly Val Glu Ser Gln Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile Asn Arg Gln Val Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly Glu His Val Phe Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu G1u Leu Lys Pro Lys Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys 485 490 4~a5 Ile Ser Glu Glu Cys Ile Ala Gln Trp Lys C~ln Thr Asn Phe Met Thr Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser Leu Glu His His His His His His <210> 3 <211> 9682 <212> DNA
<213> Artificial Sequence <220>
<221> CDS
<222> (3428)..(4543) <220>
<223> Description of Artificial Sequence: human mini TyrRS in pET20B

<400> 3 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 CdgCgtgaCC gCtdCdCttg CCdgCgCCCt agCgCCCgCt CCtttCgCtt tCttCCCttC 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgcc_ct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctatt.gg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaat:aa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatac:ac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctc~tagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccct:cc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcca gttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gac:gatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggt:ttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tat:ggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agt:gagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gac:gcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat ga~~catgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gac:cagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacatccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atc:atgcgca 3300 cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tac:gactcac 3360 tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420 tatacat atg ggg gac get ccc agc cct gaa gag aaa ctg cac ct:t atc 3469 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu His Leu Ile

10/54 acc cgg aac ctg cag gag gtt ctg ggg gaa gag aag ctg aag gag ata 3517 Thr Arg Asn Leu Gln Glu Va1 Leu Gly Glu Glu Lys Leu Lys Gl.u Ile ctg aag gag cgg gaa ctt aaa att tac tgg gga acg gca acc acg ggc 3565 Leu Lys Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly aaa cca cat gtg get tac ttt gtg ccc atg tca aag att gca gac ttc 3613 Lys Pro His Val Ala Tyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe tta aag gca ggg tgt gag gta aca att ctg ttt gcg gac ctc cac gca 3661 Leu Lys Ala Gly Cys Glu Val Thr I1e Leu Phe A1a Asp Leu His Ala tac ctg gat aac atg aaa gcc cca tgg gaa ctt cta gaa ctc cga gtc 3709 Tyr Leu Asp Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val agt tac tat gag aat gtg atc aaa gca atg ctg gag agc att ggt gtg 3757 Ser Tyr Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val r_cc ttg gag aag ctc aag ttc atc aaa ggc act gat tac cag ctc agc 3805 Pro Leu Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser aaa gag tac aca cta gat gtg tac aga ctc tcc tcc gtg gtc aca cag 3853 Lys Glu Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Va1 Val Thr Gln cac gat tce aag aag get gga get gag gtg gta aag cag gtg gag cac 3901 His Asp Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val G1u His cct ttg ctg agt ggc ctc tta tac ccc gga ctg cag get ttg gat gaa 3949 Pro Leu Leu Ser Gly Leu Leu Tyr Pro Gly Leu G1n Ala Leu Asp Glu gag tat tta aaa gta gat gcc caa ttt gga ggc att gat cag aga aag 3997 Glu Tyr Leu Lys Val Asp Ala Gln Phe Gly Gly I1e Asp Gln Arg Lys ;stt ttc acc ttt gca gag aag tac ctc cct gca ctt ggc tat tc:a aaa 4045 Ile Phe Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys 195 200 2_05 cgg gtc cat ctg atg aat cct atg gtt cca gga tta aca ggc agc aaa 4093 Arg Val His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys atg agc tct tca gaa gag gag tcc aag att gat ctc ctt gat cgg aag 4141 Met Ser Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys gag gat gtg aag aaa aaa ctg aag aag gcc ttc tgt gag cca gga aat 4189 Glu Asp Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro G1y Asn

11/54 gtggagaacaat ggggtt ctgtccttcatc aagcatgtc ctttttccc 4237 ValGluAsnAsn GlyVal LeuSerPheIle LysHisVal LeuPhePro 255 260 ~65 270 cttaagtccgag tttgtg atcctacgagat gagaaatgg ggtggaaac 9285 LeuLysSerGlu PheVal IleLeuArgAsp GluLysTrp G1yGlyAsn aaaacctacaca gettac gtggacctggaa aaggacttt getgetgag 9333 LysThrTyrThr AlaTyr ValAspLeuGlu LysAspPhe AlaAlaGlu gttgtacatcct ggagac ctgaagaattct gttgaagtc gcactgaac 4381 ValValHisPro GlyAsp LeuLysAsnSer ValGluVal AlaLeuAsn aagttgctggat ccaatc cgggaaaagttt aatacccct gccctgaaa 4429 LysLeuLeuAsp ProIle ArgGluLysPhe AsnThrPro AlaLeuLys aaaetggecagc getgce tacecagatccc t.caaagcag aagcc atg 4477 a LysLeuAlaSer AlaAla TyrProAspPro SerLysG1n LysProMet 335 340 p45 350 gccaaaggccct gccaag aattcagaacca gaggaggtc atcctcgag 4525 AlaLysGlyPro AlaLys AsnSerGluPro GluGluVal IleLeuGlu 355 360 3h5 caccaccaccac caccac tgagatccgg ctgctaacaa 4573 agcccgaaag HisHisHisHis HisHis gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct 4633 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat 4682 <210> 4 <211> 372 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: human mini TyrRS in pET20B
<400> 4 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys heu His heu Ile Thr Arg Asn Leu Gln Glu Val Leu Gly Glu Glu Lys Leu Lys Glu I1e La_u Lys Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro His Val Ala Tyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys

12/54 Ala Gly Cys Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu Asp Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Arg Val Ser Tyr 'ryr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp 'Pyr Gln Leu Ser Lys Glu Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln His Asp Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His Pro Leu Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu Giu Tyr Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Arg Lys Ile Phe Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys Glu Asp 225 230 2.35 240 Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Pro Gly Asn Val Glu Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Phe Pro Le a Lys Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp Gly Gly Asn Lys Thr Tyr Thr Ala Tyr Val Asp Leu Gl.u Lys Asp Phe Ala Ala Glu V<~1 Val His Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn Lys Leu Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Fro Met A.La Lys Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Leu Glu His His

13/54 His His His His <210> 5 <211> 4100 <212> DNA
<213> Artificial Sequence <220>
<221> CDS
<222> (3428)..(3961) <220>
<223> Description of Artificial Sequence: human TyrRS
carboxyl-terminal domain in pET20B
<400> 5 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tct:tcccttc 120 ctttctcgcr_ acgttcgccg gctttccccg tcaagctcta aatcgggggc tcc:ctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agt:ccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga ca~icgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctc:gccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ct<a agcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt g~~aggaccac ttctgcgctc 1260

14/59 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagac:ag atcgctgaga taggtgcctc 1490 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgt:ca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttc cgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgi~agttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2090 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatcttt=at agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggcctttt:gc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aag cggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tc<~gggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg tt<~agggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat ga<icatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000

15/54 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgt:at cggtgattca ttc;tgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360 tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420 tatacat atg cca gag gag gtc atc cca tcc cgg ctg gat atc cgt gtg 3469 Met Pro Glu Glu Val Ile Pro Ser Arg Leu Asp Ile Arg Val ggg aaa atc atc act gtg gag aag cac cca gat gca gac agc ctg tat 3517 Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala Asp Ser Leu Tyr gta gag aag att gac gtg ggg gaa get gaa cca cgg act gtg gtg agc 3565 Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg Thr Val Val Ser ggc ctg gta cag ttc gtg ccc aag gag gaa ctg cag gac agg ctg gta 3613 Gly Leu Val Gln Phe Val Pro Lys Glu Glu Leu Gln Asp Arg Leu Val gtg gtg ctg tgc aac ctg aaa ccc cag aag atg aga gga gtc g<~g tcc 3661 Val Val Leu Cys Asn Leu Lys Pro Gln Lys Met Arg Gly Val G_Lu Ser caa gge atg ett ctg tgt get tct ata gaa ggg ata aac cgc crag gtt 3709 Gln Gly Met Leu Leu Cys Ala Ser Ile Glu C~ly Ile Asn Arg Gln Val gaa cct ctg gac cct ccg gca ggc tct get cct ggt gag cac gtg ttt 3757 Glu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly Glu His Val Phe gtg aag ggc tat gaa aag ggc caa cca gat gag gag ctc aag ccc aag 3805 Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu Leu Lys Pro Lys 115 120 l:?5 aag aaa gte tte gag aag ttg cag get gac tte aaa att tet gag gag 3853 Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys Ile Ser Glu Glu tgc atc gca cag tgg aag caa acc aac ttc atg acc aag ctg ggc tcc 3901 Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr Lys Leu Gly Ser att tcc tgt aaa tcg ctg aaa ggg ggg aac att agc ctc gag cac cac 3949 Ile Ser Cys Lys Ser Leu Lys Gl.y Gly Asn Ile Ser Leu Glu His His cac cac cac cac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt 4001

16/54 His His His His tggctgctgc caccgctgag caataactag cataacccct tggggcctct aaacgggtct 4061 tgaggggttt tttgctgaaa ggaggaacta tatccggat 4100 <210> 6 <211> 178 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: human TyrRS
carboxyl-terminal domain in pET20B
<400> 6 Met Pro Glu Glu Val Ile Pro Ser Arg Leu Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala Asp Ser Leu Tyr Val Glu Lys Ile Asp Val Gly Glu Ala Glu Pro Arg Thr Val Val Ser G:Ly Leu Val Gln Phe Val Pro Lys Glu Glu Leu Gln Asp Arg Leu Val Val Val Leu Cys Asn Leu Lys Pro Gln Lys Met Arg Gly Val Glu Ser G.Ln Gly Met Leu Leu Cys Ala Ser Ile Glu Gly Ile Asn Arg Gln Val G_Lu Pro Leu Asp Pro Pro Ala Gly Ser Ala Pro Gly C~lu His Val Phe Val Lys Gly Tyr Glu Lys Gly Gln Pro Asp Glu Glu Leu Lys Pro Lys Lys Lys Val Phe Glu Lys Leu Gln Ala Asp Phe Lys Ile Ser G1u Glu Cys Ile Ala Gln Trp Lys Gln Thr Asn Phe Met Thr Lys Leu Gly Ser Ile Ser Cys Lys Ser Leu Lys Gly Gly Asn Ile Ser Leu Glu His His His His 165 170 1'75 His His <210> 7 <211> 4682 <212> DNA
<213> Artificial Sequence

17/54 <220>
<221> CDS
<222> (3428)..(4543) <220>
<223> Description of Artificial Sequence: human mini TyrRS mutant in pET20B
<400> 7 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 290 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agi~ccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caa atatgta 540 tccgctcatg agacaataac cctgataaat gcttcaat:aa tattgaaaaa gga agagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500

18/54 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atc~tcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatct.gc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggt:ttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tat=ggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ct<:acatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2960 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac act=ccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgt:at cggtgattca ttctgctaac 3290

19/54 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360 tatagggaga ccacaacggt ttccctctag aaataatt=tt gtttaacttt aag aaggaga 3420 tatacat atg ggg gac get ece age cct gaa gag aaa ctg cac c~t atc 3469 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu His Le a I1_e acccggaacctg caggag gttctgggg gaagagaagctg aaggagata 3517 ThrArgAsnLeu GlnGlu ValLeuGly GluGluLysLeu LysGluIle ctgaaggagcgg gaactt aaaatttac tggggaacggca accacgggc 3565 LeuLysGluArg GluLeu LysIleTyr TrpGlyThrAla ThrThrGly aaaecacatgtg gettac tttgtgece atgtcaaagatt gcagactte 3613 LysProHisVal AlaTyr PheValPro MetSerLysIle AlaAspPhe ttaaaggcaggg tgtgag gtaacaatt ctgtttgcggac ctccacgca 3661 LeuLysAlaGly CysGlu ValThrIle LeuPheAlaAsp LeuH:isAla tacctggataac atgaaa gccccatgg gaacttctagaa ctgcaggtc 3709 TyrLeuAspAsn MetLys AlaProTrp Glul,euLeuGlu LeuG.LnVal agttactatgag aatgtg atcaaagca atgctggagagc attggtgtg 3757 SerTyrTyrGlu AsnVal IleLysAla MetLeuGluSer IleGlyVal cccttggagaag ctcaag ttcatcaaa ggcactgattac cagctcagc 3805 ProLeuGluLys LeuLys PheIleLys GlyThrAspTyr GlnLe Ser a 115 120 1'?5 aaagagtacaca ctagat gtgtacaga ctct:cctccgtg gtcacacag 3853 LysGluTyrThr LeuAsp ValTyrArg LeuSerSerVal ValThrGln cacgattccaag aagget ggagetgag gtggtaaagcag gtggagcac 3901 HisAspSerLys LysAla GlyAlaGlu ValValLysGln ValGluHis cctttgetgagt ggecte ttatacece ggaetgcagget ttggatgaa 3949 ProLeuLeuSer GlyLeu LeuTyrPro GlyLeuGlnAla LeuAspGlu gagtatttaaaa gtagat gcccaattt ggacxgcattgat cagagaaag 3997 GluTyrLeuLys Va1Asp AlaGlnPhe GlyGlyIleAsp GlnArgLys attttcaccttt gcagag aagtacctc cctgcacttggc tattcaaaa 4045 IlePheThrPhe AlaGlu LysTyrLeu ProAlaLeuGly TyrSerLys cgggtccatctg atgaat cctatggtt ccaggattaaca ggcagcaaa 4093

20/54 Arg Val His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys atg agc tct tca gaa gag gag tcc aag att gat ctc ctt gat cgg aag 4141 Met Ser Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp A:rg Lys gag gat gtg aag aaa aaa ctg aag aag gcc ttc tgt gag cca gga aat 4189 Glu Asp Val Lys Lys Lys Leu Lys Lys A1a Phe Cys G1u Pro G-Ly Asn gtg gag aac aat ggg gtt ctg tcc ttc atc aag cat gtc ctt ttt ccc 9237 Val Glu Asn Asn Gly Val Leu Ser Phe Ile Lys His Val Leu Fhe Pro ctt aag tcc gag ttt gtg atc cta cga gat gag aaa tgg ggt gg a aac 9285 Leu Lys Ser Glu Phe Val Ile Leu Arg Asp Glu Lys Trp G1y Gly Asn aaa acc tac aca get tac gtg gac ctg gaa aag gac ttt get get gag 9333 Lys Thr Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu gtt gta cat cct gga gac ctg aag aat tct gtt gaa gtc gca ctg aac 4381 Val Val His Pro Gly Asp Leu Lys Asn Ser Val Glu Val Ala Leu Asn aag ttg ctg gat cca atc cgg gaa aag ttt aat acc cct gcc ctg aaa 4429 Lys Leu Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys aaa etg gce age get gcc tac cca gat ece tca aag cag aag cca atg 4477 Lys Leu Ala Ser Ala Ala Tyr Pro Asp Pro :~er Lys Gln Lys P:ro Met gcc aaa ggc cct gcc aag aat tca gaa cca gag gag gtc atc ctc gag 4525 Ala Lys Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Le a Glu cac cac cac cac cac cac tgagatccgg ctgctaacaa agcccgaaag 4573 His His His His His His gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct 4633 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat 4682 <210> 8 <211> 372 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: human mini TyrRS mutant in pET20B
<400> 8 Met Gly Asp Ala Pro Ser Pro Glu Glu Lys Leu His Leu Ile Thr Arg

21/54 i 5 10 15 Asn Leu Gln Glu Val Leu Gly Glu Glu Lys Leu Lys Glu Ile Leu Lys Glu Arg Glu Leu Lys Ile Tyr Trp Gly Thr Ala Thr Thr Gly Lys Pro His Val Ala Tyr Phe Val Pro Met Ser Lys Ile Ala Asp Phe Leu Lys Ala Gly Cys Glu Val Thr Ile Leu Phe Ala Asp Leu His Ala Tyr Leu Asp Asn Met Lys Ala Pro Trp Glu Leu Leu Glu Leu Gln Val Ser Tyr Tyr Glu Asn Val Ile Lys Ala Met Leu Glu Ser Ile Gly Val Pro Leu Glu Lys Leu Lys Phe Ile Lys Gly Thr Asp Tyr Gln Leu Ser Lys Giu Tyr Thr Leu Asp Val Tyr Arg Leu Ser Ser Val Val Thr Gln His Asp Ser Lys Lys Ala Gly Ala Glu Val Val Lys Gln Val Glu His Pro Leu Leu Ser Gly Leu Leu Tyr Pro Gly Leu Gln Ala Leu Asp Glu G1u Tyr Leu Lys Val Asp Ala Gln Phe Gly Gly Ile Asp Gln Arg Lys I1e Phe Thr Phe Ala Glu Lys Tyr Leu Pro Ala Leu Gly Tyr Ser Lys Arg Val His Leu Met Asn Pro Met Val Pro Gly Leu Thr Gly Ser Lys Met Ser Ser Ser Glu Glu Glu Ser Lys Ile Asp Leu Leu Asp Arg Lys G1u Asp 225 230 ?35 240 Val Lys Lys Lys Leu Lys Lys Ala Phe Cys Glu Fro Gly Asn Val Glu Asn Asn Gly Val Leu Ser Phe Ile Lys His 'Jal Leu Phe Pro Leu Lys Ser Glu Phe Val Ile Leu Arg Asp Glu Lys 'Trp Gly Gly Asn Lys Thr Tyr Thr Ala Tyr Val Asp Leu Glu Lys Asp Phe Ala Ala Glu Val Val His Pro Gly Asp Leu Lys Asn Ser Val Glu 'Jal A1a Leu Asn Lys Leu

22/54 Leu Asp Pro Ile Arg Glu Lys Phe Asn Thr Pro Ala Leu Lys Lys Leu Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met A.La Lys Gly Pro Ala Lys Asn Ser Glu Pro Glu Glu Val Ile Leu Glu His His His His His His <210> 9 <211> 5018 <212> DNA
<213> Artificial Sequence <220>
<221> CDS
<222> (3428)..(4879) <220>
<223> Description of Artificial Sequence: human full-length TrpRS in pET20B
<400> 9 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttt.tc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gc~Jttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt tt~~gccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 890 tattgacgcc gggcaagagc aactcggtcg ccgcatac,ac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960

23/54 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caa cgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac tt~~tgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caa aaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtccat ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc cca gcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760

24/59 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt cca cagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct ga~~ttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3290 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360 tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420 tatacat atg ccc aac agt gag ccc gca tct c~tg ctg gag ctg ttc aac 3469 Met Pro Asn Ser Glu Pro Ala Ser Leu Leu Glu Leu Phe Asn agcatcgccaca caaggg gagctcgta aggtccctc aaagcgggaaat 3517 SerIleAlaThr GlnGly GluLeuVal ArgSerLeu LysAlaGlyAsn gcgtcaaaggat gaaatt gattctgca gtaaagatg ttggtgtcatta 3565 AlaSerLysAsp GluIle AspSerAla ValI,ysMet LeuValSerLeu aaaatgagctac aaaget gccgegggg gagslattac aaggetga tgt 3613 c LysMetSerTyr LysAla AlaAlaGly Gl.uAspTyr LysAlaAspCys cctccagggaac ccagca cctaccagt aatcatggc ccagatgccaca 3661 ProProGlyAsn ProAla ProThrSer AsnHisGly ProAspA1aThr gaagetgaagag gatttt gtggaccca tgg<icagta cagacaagcagt 3709 GluAlaGluGlu AspPhe ValAspPro Trp'?'hrVal GlnThrSerSer gcaaaaggcata gactac gataagctc attgttcgg tttggaagtagt 3757 AlaLysGlyIle AspTyr AspLysLeu IleValArg FheGlySerSer aaaattgacaaa gagcta ataaaccga atagagaga gccaccggccaa 3805 LysIleAspLys GluLeu IleAsnArg I1eGluArg A1aThrG1yGln agaccacaccac ttcctg cgcagaggc atcttcttc tcacacagagat 3853 ArgProHisHis PheLeu ArgArgG1y IleFheFhe SerHisArgAsp

25/54 atg aat cag gtt ctt gat gcc tat gaa aat aag aag cca ttt tat ctg 3901 Met Asn Gln Val Leu Asp Ala Tyr Glu Asn I~ys Lys Pro Phe Tyr Leu tac acg ggc cgg ggc ccc tct tct gaa gca atg cat gta ggt cac ctc 3949 Tyr Thr Gly Arg Gly Pro Ser Ser Glu Al.a Met His Val Gly H.is Leu att cca ttt att ttc aca aag tgg ctc cag gat gta ttt aac gtg ccc 3997 Ile Pro Phe Ile Phe Thr Lys Trp Leu Gl.n Asp Va1 Phe Asn Val Pro ttg gtc atc cag atg acg gat gac gag aag tat ctg tgg aag gac ctg 4095 Leu Val Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu Trp Lys Asp Leu acc ctg gac cag gcc tat ggc gat get gtt gag aat gcc aag gac atc 4093 Thr Leu Asp Gln Ala Tyr Gly Asp Ala Val Glu Asn Ala Lys Asp Ile atc gcc tgt ggc ttt gac atc aac aag act ttc ata ttc tct gac ctg 9141 Ile Ala Cys Gly Phe Asp Ile Asn Lys Thr l?he Ile Phe Ser Asp Leu gac tac atg ggg atg agc tca ggt ttc tac aaa aat gtg gtg aag att 4189 Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Val Val Lys I1e caa aag cat gtt acc ttc aac caa gtg aaa ggc att ttc ggc ttc act 4237 Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Ile Phe Gly Phe Thr gac agc gac tge att ggg aag ate agt ttt cet gec ate cag get get 4285 Asp Ser Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala Ile Gln Ala Ala ccc tcc ttc agc aac tca ttc cca cag atc ttc cga gac agg acg gat 4333 Pro Ser Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg Thr Asp atc cag tgc ctt atc cca tgt gcc att gac cag gat cct tac ttt aga 4381 Ile Gln Cys Leu Ile Pro Cys Ala Ile Asp Gl.n Asp Pro Tyr Phe Arg atg aca agg gac gtc gcc ccc agg atc ggc tat cct aaa cca gcc ctg 4429 Met Thr Arg Asp Val Ala Pro Arg Ile Gly Tyr Fro Lys Pro Ala Leu ttg cac tcc acc ttc ttc cca gcc ctg cag ggc gcc cag acc aaa atg 4477 Leu His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln Thr Lys Met agt gcc agc gac cca aac tcc tcc atc ttc ctc acc gac acg gcc aag 4525 Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr Asp Thr Ala Lys cag atc aaa acc aag gtc aat aag cat gcg ttt tct gga ggg aga gac 4573 Gln Ile Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly Gly Arg Asp

26/54 acc atc gag gag cac agg cag ttt ggg ggc aac tgt gat gtg gac gtg 4621 Thr Ile Glu Glu His Arg G1n Phe Gly Gly Asn Cys Asp Val A.sp Val tct ttc atg tac ctg acc ttc ttc ctc gag gac gac gac aag ctc gag 4669 Ser Phe Met Tyr Leu Thr Phe Pile Leu Glu Asp Asp Asp Lys Leu Glu cag atc agg aag gat tac acc agc gga gcc atg ctc acc ggt gag ctc 4717 Gln Ile Arg Lys Asp Tyr Thr Ser Gly Ala Met Leu Thr Gly Glu Leu aag aag gca ctc ata gag gtt ctg cag ccc t~tg atc gca gag cac cag 4765 Lys Lys Ala Leu I1e Glu Val Leu Gln Pro Leu Ile Ala Glu His Gln gcc cgg cgc aag gag gtc acg gat gag ata gtg aaa gag ttc atg act 4813 Ala Arg Arg Lys Glu Val Thr Asp G1u Ile Val Lys Glu Phe Met Thr ccc cgg aag ctg tcc ttc gac ttt cag aag ctt gcg gcc gca ctc gag 4861 Pro Arg Lys Leu Ser Phe Asp Phe Gln Lys Leu Ala Ala Ala Leu Glu cac cac cac cac cac cac tgagatccgg ctgctaacaa agcccgaaag 4909 His His His His His His gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct 4969 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat 5018 <210> 10 <211> 484 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: human full-length TrpRS in pET20B
<400> 10 Met Pro Asn Ser Glu Pro Ala Ser Leu Leu Glu Leu Phe Asn Ser Ile Ala Thr Gln Gly Glu Leu Val Arg Ser Leu Lys Ala Gly Asn Ala Ser Lys Asp Glu Ile Asp Ser Ala Val Lys Met Leu Val Ser Leu Lys Met Ser Tyr Lys Ala Ala Ala Gly Glu Asp Tyr Lys Ala Asp Cys Pro Pro Gly Asn Pro Ala Pro Thr Ser Asn His Gly Pro Asp Ala Thr Glu Ala

27/54 Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr Ser Ser A1a Lys 85 90 a5 Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg A1a Thr Gly Gln Arg Pro His His Phe Leu Arg Arg Gly I1e Phe Phe Ser His Arg Asp Met Asn Gln Val Leu Asp Ala Tyr Glu Asn Lys Lys Pro Phe Tyr Leu Tyr Thr Gly Arg Gly Pro Ser Ser Glu Ala Met His Val Gly His Leu Ile Pro Phe Ile Phe Thr Lys Trp Leu Gln Asp Val Phe Asn Val Pro Leu Val Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu Trp Lys Asp Leu Thr Leu Asp Gln Ala Tyr Gly Asp Ala Val Glu Asn Ala Lys Asp Ile Ile Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile Phe Ser Asp Leu Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Val Val Lys Ile Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Iie Phe Gly Phe Thr Asp Ser Asp Cys I1e Gly Lys Ile Ser Phe Pro Ala Ile Gln Ala Ala Pro Ser Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg Thr Asp Ile Gln Cys Leu Ile Pro Cys Ala Ile Asp Gln Asp Pro Tyr Phe Arg Met Thr Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro Lys Pro Ala Leu Leu His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln Thr Lys Met Ser Ala Ser Asp Pro Asn Ser Ser I1e Phe Leu Thr Asp Thr Ala Lys Gln Ile Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly Gly Arg Asp Thr Ile Glu Glu His Arg Gln Phe Gly G1y Asn Cys Asp Val Asp Val Ser Phe

28/54 Met Tyr Leu Thr Phe Phe Leu G1u Asp Asp Asp Lys Leu Glu Gln Ile Arg Lys Asp Tyr Thr Ser Gly Ala Met Leu 'rhr Gly Glu Leu Lys Lys Ala Leu Ile Glu Val Leu Gln Pro Leu Ile Ala Glu His Gln Ala Arg Arg Lys Glu Val Thr Asp Glu Ile Val Lys Glu Phe Met Thr Pro Arg Lys Leu Ser Phe Asp Phe Gln Lys Leu Ala A1a Ala Leu Glu His His His His His His <210> 11 <211> 4877 <212> DNA
<213> Artificial Sequence <220>
<221> CDS
<222> (3428)..(4738) <220>
<223> Description of Artificial Sequence: human mini TrpRS in pET20B
<400> 11 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct t:tgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg to aaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780

29/54 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt caagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ca atgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520

30/54 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tc~~agctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggg gg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca t.gttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3290 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360 tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3920 tatacat atg agc tac aaa get gcc gcg ggg gag gat tac aag get gac 3469 Met Ser Tyr Lys Ala Ala Ala Gly Glu Asp Tyr Lys Ala Asp tgt cct cca ggg aac cca gca cct acc agt aat cat ggc cca gat gcc 3517 Cys Pro Pro Gly Asn Pro Ala Pro Thr Ser Asn His Gly Pro Asp Ala aca gaa get gaa gag gat ttt gtg gac cca tgg aca gta cag aca agc 3565 Thr Glu Ala Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr Ser agt gca aaa ggc ata gac tac gat aag ctc att gtt cgg ttt gga agt 3613 Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser agt aaa att gac aaa gag cta ata aac cga ata gag aga gcc acc ggc 3661 Ser Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr Gly caa aga cca cac cac ttc ctg cgc aga ggc atc t.tc ttc tca cac aga 3709 Gln Arg Pro His His Phe Leu Arg Arg Gly Ile Phe Phe Ser His Arg gat atg aat cag gtt ctt gat gcc tat gaa aat aag aag cca ttt tat 3757 Asp Met Asn Gln Val Leu Asp Ala Tyr Glu Asn hys Lys Pro Phe Tyr ctg tac acg ggc cgg ggc ccc tct tct gaa gca atg cat gta ggt cac 3805

31/54 LeuTyrThr GlyArgGly ProSerSer GluA1aMet HisValGly His ctcattcca tttattttc acaaagtgg ctccaggat gtatttas gtg 3853 c LeuIlePro PheIlePhe ThrLysTrp LeuGlnAsp ValPheAsn Val cccttggtc atccagatg acggatgac gagaagtat ctgtggaag gac 3901 ProLeuVal IleGlnMet ThrAspAsp GluLysTyr heuTrpLys Asp etgaccetg gaccaggce tatggegat getgttgag aatgceaag gac 3949 LeuThrLeu AspGlnAla TyrGlyAsp AlaValGlu AsnAlaLys Asp atcatcgcc tgtggcttt gacatcaac aagactttc atattct.ctgac 3997 IleIleAla CysGlyPhe AspIleAsn LysThrPhe IlePheSer Asp ctggactac atggggatg agctcaggt ttctacaaa aatgtggtg aag 4045 LeuAspTyr MetGlyMet SerSerGly PheTyrLys AsnValVal Lys attcaaaag catgttacc ttcaaccaa gtga ggc attttcggc ttc 4093 as IleGlnLys HisValThr PheAsnGln Val~~ysGly IlePheGly Phe 210 2.15 220 actgacagc gactgcatt gggaagatc agttttcet gecatecag get 4141 ThrAspSer AspCysIle GlyLysIle SerPhePro AlaIleGln Ala getecctcc ttcageaac tcattecca cagatctte cgagacagg acg 4189 AlaProSer PheSerAsn SerPhePro GlnIlePhe ArgAspArg Thr gatatccag tgccttatc ccatgtgcc attgaccag gatccttac ttt 4237 AspIleGln CysLeuIle ProCysAla IleAspGln AspProTyr Phe agaatgaca agggacgtc gcccccagg atcggctat cctaaacca gcc 4285 ArgMetThr ArgAspVal AlaProArg IleGlyTyr ProLysPro Ala ctgttgcac tccaccttc ttcccagcc ctgcagggc gcccagacc aaa 4333 LeuLeuHis SerThrPhe PheProAla LeuC,lnGly AlaGlnThr Lys atgagtgcc agcgaccca aactcctcc atcttcctc accgacacg gcc 4381 MetSerAla SerAspPro AsnSerSer IlePheLeu ThrAspThr A1a aagcagatc aaaaccaag gtcaataag catgcgttt tctggaggg aga 4429 LysGlnIle LysThrLys Va1AsnLys HisAlaPhe SerGlyGly Arg gacaccatc gaggagcac aggcagttt gggggcaac tgtgatgtg gac 4477 AspThrIle GluGluHis ArgGlnPhe GlyGlyAsn CysAspVal Asp

32/54 gtg tct ttc atg tac ctg acc ttc ttc ctc gag gac gac gac aag ctc 4525 Val Ser Phe Met Tyr Leu Thr Phe Phe Leu Glu Asp Asp Asp Lys Leu gag cag atc agg aag gat tac acc agc gga gcc atg ctc acc ggt gag 4573 Glu Gln Ile Arg Lys Asp Tyr Thr Ser Gly A1a Met L,eu Thr G1y G1u ctc aag aag gca ctc ata gag gtt ctg cag ccc ttg atc gca gag cac 4621 Leu Lys Lys Ala Leu Ile Glu Val Leu Gln Pro Leu Ile Ala Glu His cag gcc cgg cgc aag gag gtc acg gat gag ata gtg aaa gag ttc atg 4669 Gln Ala Arg Arg Lys Glu Val Thr Asp Glu Il.e Val hys Glu Phe Met act ccc cgg aag ctg tcc ttc gac ttt cag aag ctt gcg gcc gca ctc 4717 Thr Pro Arg Lys Leu Ser Phe Asp Phe Gln Lys Leu Ala Ala Ala Leu gag cac cac cac cac cac cac tgagatccgg ctgctaacaa agcccgaaag 4768 Glu His His His His His His gaagctgagt tggctgctgc caccgctgag caataactag cataacccct tggggcctct 4828 aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat 4877 <210> 12 <211> 437 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: human mini TrpRS in pET20B
<400> 12 Met Ser Tyr Lys Ala Ala Ala Gly Glu Asp Tyr hys Ala Asp Cys Pro Pro Gly Asn Pro Ala Pro Thr Ser Asn His Gly Pro Asp Ala Thr Glu Ala Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr Ser Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg Pro His His Phe Leu Arg Arg Gly Ile Phe Phe Ser His Arg Asp Met Asn Gln Val Leu Asp Ala Tyr Glu Asn Lys Lys Pro Phe Tyr Leu Tyr

33/59 Thr Gly Arg Gly Pro Ser Ser Glu Ala Met His Val Gly His Leu Ile Pro Phe Ile Phe Thr Lys Trp Leu Gln Asp Val Phe Asn Val Pro Leu Val Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu Trp Lys Asp Leu Thr Leu Asp Gln Ala Tyr Gly Asp Ala Val Glu Asn Ala Lys Asp Ile Ile Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe I1e Phe Ser Asp Leu Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Va1 Val Lys I1e Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Ile Phe Gly Phe 'I'hr Asp Ser Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala Ile Gln Ala Ala Pro 225 230 ?35 240 Ser Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg Thr Asp I.le Gln Cys Leu Ile Pro Cys Ala I1e Asp Gln Asp Pro Tyr Phe Arg Met Thr Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro Lys Pro Ala Leu Leu His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln Thr Lys Met Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr P.sp Thr Ala Lys Gln Ile Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly Gly Arg Asp Thr Ile Glu Glu His Arg Gln Phe Gly Gly Asn Cys Asp Val Asp Val Ser Phe Met Tyr Leu Thr Phe Phe Leu Glu Asp Asp Asp Lys Leu Glu Gln Ile Arg Lys Asp Tyr Thr Ser Gly Ala Met Leu Thr Gly Glu Leu Lys 370 375 :~80 Lys Ala Leu Ile Glu Val Leu Gln Pro Leu Ile Ala Glu His Gln Ala Arg Arg Lys Glu Val Thr Asp Glu Ile Val Lys Glu Phe Met Thr Pro Arg Lys Leu Ser Phe Asp Phe Gln Lys Leu Ala Ala Ala Leu Glu His

34/54 His His His His His <210> 13 <211> 4811 <212> DNA
<213> Artificial Sequence <220>
<221> CDS
<222> (3428)..(4672) <220>
<223> Description of Artificial Sequence: human supermini TrpRS in pET20B
<400> 13 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 590 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccct:ttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200

35/54 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttacl=ca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa ga~~gatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtca<~gg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2990

36/54 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccatt=ca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgt:at cggtgattca ttctgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaa<:ga caggagcacg atcatgcgca 3300 cccgtggcca ggacccaacg ctgcccgaga tctcgatc:cc gcgaaattaa tac~gactcac 3360 tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420 tatacat atg agt aat cat ggc cca gat gcc aca gaa get gaa gag gat 3469 Met Ser Asn His Gly Pro Asp Ala Thr Glu Ala Glu G:Lu Asp ttt gtg gac cca tgg aca gta cag aca agc agt gca aaa ggc ata gac 3517 Phe Val Asp Pro Trp Thr Val Gln Thr Ser Ser Ala Lys Gly I:Le Asp tac gat aag ctc att gtt cgg ttt gga agt agt aaa att gac aaa gag 3565 Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser Ser Lys Ile Asp Lys Glu cta ata aac cga ata gag aga gcc acc ggc caa aga cca cac cac ttc 3613 Leu Ile Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg Fro His His Phe ctg cgc aga ggc atc ttc ttc tca cac aga gat atg aat cag gtt ctt 3661 Leu Arg Arg Gly Ile Phe Phe Ser His Arg Asp Met Asn Gln Val Leu gat gcc tat gaa aat aag aag cca ttt tat ctg tac acg ggc cgg ggc 3709 Asp Ala Tyr Glu Asn Lys Lys Pro Phe Tyr heu Tyr Thr Gly Arg Gly ccc tct tct gaa gca atg cat gta ggt cac ctc att cca ttt at=t ttc 3757 Pro Ser Ser Glu Ala Met His Val Gly His I~eu Ile Pro Phe Ile Phe 95 100 7.05 110 aca aag tgg ctc cag gat gta ttt aac gtg ccc ttg gtc atc cag atg 3805 Thr Lys Trp Leu Gln Asp Val Phe Asn Val Pro Leu Val Ile Gln Met 115 120 l:?5 acg gat gac gag aag tat ctg tgg aag gac r_tg acc ctg gac cag gcc 3853 Thr Asp Asp Glu Lys Tyr Leu Trp Lys Asp Leu Thr Leu Asp Gln Ala tat ggc gat get gtt gag aat gcc aag gac atc atc gcc tgt ggc ttt 3901 Tyr Gly Asp Ala Val Glu Asn Ala Lys Asp I:le Ile Ala Cys Gly Phe gac atc aac aag act ttc ata ttc tct gac ctg gac tac atg ggg atg 3999 Asp Ile Asn Lys Thr Phe Ile Phe Ser Asp Leu Asp Tyr Met Gly Met

37/54 agc tca ggt ttc tac aaa aat gtg gtg aag att caa aag cat gtt acc 3997 Ser Ser Gly Phe Tyr Lys Asn Val Val Lys Ile Gln Lys His Val Thr 175 180 1.85 190 ttc aac caa gtg aaa ggc att ttc ggc ttc act gac agc gac tgc att 4045 Phe Asn Gln Val Lys Gly Ile Phe Gly Phe Thr Asp Ser Asp Cys Ile ggg aag atc agt ttt cct gcc atc cag get get ccc tcc ttc agc aac 4093 Gly Lys Ile Ser Phe Pro Ala Ile Gln Ala Ala Pro Ser Phe Ser Asn tca ttc cca cag atc ttc cga gac agg acg gat atc cag tgc c't atc 4141 Ser Phe Pro Gln Ile Phe Arg Asp Arg Thr Asp Ile Gln Cys Leu Ile cca tgt gcc att gac cag gat cct tac ttt aga atg aca agg gac gtc 4189 Pro Cys Ala Ile Asp Gln Asp Pro Tyr Phe Arg Met Thr Arg Asp Val gcc ccc agg atc ggc tat cct aaa cca gcc ctg ttg cac tcc acc ttc 4237 Ala Pro Arg Ile Gly Tyr Pro Lys Pro Ala Leu Leu His Ser Thr Phe ttc cca gcc ctg cag ggc gcc cag acc aaa atg agt gcc agc ga c cca 4285 Phe Pro Ala Leu Gln Gly Ala Gln Thr Lys Met Ser A1a Ser Asp Pro aac tcc tcc atc ttc ctc acc gac acg gcc aag cag atc aaa acc aag 4333 Asn Ser Ser Ile Phe Leu Thr Asp Thr Ala Lys Gln Ile Lys Thr Lys gtc aat aag cat gcg ttt tct gga ggg aga gac acc atc gag gag cac 9381 Val Asn Lys His Ala Phe Ser Gly G1y Arg Asp Thr I1e G1u G:Lu His agg cag ttt ggg ggc aac tgt gat gtg gac gtg tct ttc atg tac ctg 9429 Arg Gln Phe Gly Gly Asn Cys Asp Val Asp Val Ser Phe Met Tyr Leu acc ttc ttc ctc gag gac gac gac aag ctc gag cag atc agg aag gat 4477 Thr Phe Phe Leu Glu Asp Asp Asp Lys Leu Glu Gln Ile Arg Lys Asp tac acc agc gga gcc atg ctc acc ggt gag ctc aag aag gca ctc ata 9525 Tyr Thr Ser Gly Ala Met Leu Thr Gly Glu I,eu Lys Lys Ala Leu Ile gag gtt ctg cag ccc ttg atc gca gag cac cag gcc cgg cgc aag gag 9573 G1u Val Leu G1n Pro Leu Ile Ala Glu His C~ln Ala Arg Arg Lys Gl.u gtc acg gat gag ata gtg aaa gag ttc atg act ccc cgg aag ctg tcc 4621 Val Thr Asp Glu Ile Val Lys Glu Phe Met Thr Pro Arg Lys Leu Ser ttc gac ttt cag aag ctt gcg gcc gca ctc gag cac cac cac cac cac 4669 Phe Asp Phe Gln Lys Leu Ala Ala Ala Leu Glu His His His H.is His

38/59 cac tgagatccgg ctgctaacaa agcccgaaag gaagctgagt tggctgctgc 4722 His caccgctgag caataactag cataacccct tggggcct:ct aaacgggtct tgaggggttt 4782 tttgctgaaa ggaggaacta tatccggat 4811 <210> 14 <211> 415 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: human supermini TrpRS in pET20B
<400> 14 Met Ser Asn His Gly Pro Asp Ala Thr Glu Ala Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr Ser Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser Ser Lys lle Asp Lys Glu Leu I1e Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg Pro His His Phe Leu Arg Arg Gly Ile Phe Phe Ser His Arg Asp Met Asn Gln Val Leu Asp Ala Tyr Glu Asn Lys Lys Pro Phe Tyr Leu Tyr Thr Gly Arg Gly Pro Ser 85 90 ~~5 Ser Glu Ala Met His Val Gly His Leu Ile Pro Phe Ile Phe Thr Lys Trp Leu Gln Asp Val Phe Asn Val Pro Leu Val Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu Trp Lys Asp Leu Thr Leu Asp Gln Ala Tyr Gly Asp Ala Val Glu Asn Ala Lys Asp Ile Ile Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile Phe Ser Asp Leu Asp '1'yr Met Gly Met Ser Ser 165 170 1'75 Gly Phe Tyr Lys Asn Val Val Lys Ile Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Ile Phe Gly Phe Thr Asp Ser Asp Cys I1e G:Ly Lys

39/54 Ile Ser Phe Pro Ala Ile Gln Ala Ala Pro Ser Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg Thr Asp Ile Gln Cys Leu Ile Pro Cys Ala Ile Asp Gln Asp Pro Tyr Phe Arg Met Thr Arg Asp Val ALa Pro 245 250 2:55 Arg Ile Gly Tyr Pro Lys Pro Ala Leu Leu His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala Gln Thr Lys Met Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr Asp Thr Ala Lys Gln -Ile Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly Gly Arg Asp Thr Ile Glu Glu His Arg Gln Phe Gly Gly Asn Cys Asp Val Asp Val Ser Phe Met Tyr Leu Thr Phe Phe Leu Glu Asp Asp Asp Lys Leu Glu Gln Ile Arg Lys Asp Tyr Thr Ser Gly Ala Met Leu Thr Gly Glu Leu Lys Lys Ala Leu I1e Glu Val Leu Gln Pro Leu Ile Ala Glu His Gln Ala Arg Arg Lys Glu Val Thr Asp Glu Ile Val Lys Glu Phe Met Thr Pro Arg Lys Leu Ser Phe Asp Phe Gln Lys Leu Ala Ala Ala Leu Glu His His His His His H:is <210> 15 <211> 4742 <212> DNA
<213> Artificial Sequence <220>
<221> CDS
<222> (3428)..(4603) <220>
<223> Description of Artificial Sequence: human inactive TrpRS in pET20B
<400> 15 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120

40/59 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctatt~gg ttaaaaaatg ag~~tgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 590 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttact.tctga caa cgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ct~~gccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1490 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920

41/54 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca tt~~tgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaar_ga caggagcacg atcatgcgca 3300 cccgtggcca ggacccaacg ctgcccgaga tctcgatccc gcgaaattaa tacgactcac 3360 tatagggaga ccacaacggt ttccctctag aaataatttt gtttaacttt aagaaggaga 3420 tatacat atg agt gca aaa ggc ata gac tac gat aag ctc att gtt cgg 3469 Met Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg ttt gga agt agt aaa att gac aaa gag cta ata aac cga ata gag aga 3517 Phe Gly Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg

42/59 gcc acc ggc caa aga cca cac cac ttc ctg cgc aga ggc atc ttc ttc 3565 Ala Thr Gly Gln Arg Pro His His Phe Leu Arg Arg Gly Ile Phe Phe tca cac aga gat atg aat cag gtt ctt gat gcc tat gaa aat as g aag 3613 Ser His Arg Asp Met Asn G1n Val Leu Asp Ala Tyr Glu Asn Lys Lys cca ttt tat ctg tac acg ggc cgg ggc ccc tct tct gaa gca atg cat 3661 Pro Phe Tyr Leu Tyr Thr Gly Arg G1y Pro .'per Ser Glu Ala Met His gta ggt cac ctc att cca ttt att ttc aca aag tgg ctc cag gat gta 3709 Val Gly His Leu Ile Pro Phe Ile Phe Thr Lys Trp Leu Gln Asp Val ttt aac gtg ccc ttg gtc atc cag atg acg gat gac gag aag tat ctg 3757 Phe Asn Val Pro Leu Val Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu tgg aag gac ctg acc etg gac cag gcc tat ggc gat get gtt gag aat 3805 Trp Lys Asp Leu Thr Leu Asp Gln Ala Tyr Gly Asp Ala Val Glu Asn gcc aag gac atc atc gcc tgt ggc ttt gac atc aac aag act ttc ata 3853 Ala Lys Asp Ile Ile Ala Cys Gly Phe Asp T_le Asn Lys Thr Phe Ile ttc tct gac ctg gac tac atg ggg atg agc tca ggt ttc tac as a aat 3901 Phe Ser Asp Leu Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn gtg gtg aag att caa aag cat gtt acc ttc aac caa gtg aaa ggc att 3949 Val Val Lys Ile Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Ile ttc ggc ttc act gac agc gac tgc att ggg aag atc agt ttt cct gcc 3997 Phe Gly Phe Thr Asp Ser Asp Cys Ile Gly hys Ile Ser Phe Pro Ala atc cag get get ecc tce ttc agc aac tca ttc cca cag ate tte ega 4045 Ile Gln Ala Ala Pro Ser Phe Ser Asn Ser Phe Pro Gln Ile Plze Arg gac agg acg gat atc cag tgc ctt atc cca tgt gcc att gac cag gat 4093 Asp Arg Thr Asp Ile Gln Cys Leu Ile Pro Cys Ala Ile Asp GLn Asp cct tac ttt aga atg aca agg gac gtc gcc ccc agg atc ggc tat cct 4141 Pro Tyr Phe Arg Met Thr Arg Asp Val Ala T'ro Arg Ile Gly Tyr Pro aaa cca gcc ctg ttg cac tcc acc ttc ttc cca gcc ctg cag ggc gcc 4189 Lys Pro Ala Leu Leu His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala cag acc aaa atg agt gcc agc gac cca aac tcc tcc atc ttc ctc acc 4237 Gln Thr Lys Met Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr 255 260 ~'65 270

43/54 gacacggccaag cagatc aaaaccaag gtcaataagcatgcg ttttct 4285 AspThrAlaLys GlnIle LysThrLys ValAsnLysHisAla PheSer ggagggagagac accatc gaggagcac aggcagtttgggggc aactgt 4333 GlyGlyArgAsp ThrIle GluGluHis ArgGlnPheGlyGly AsnCys gatgtggacgtg tctttc atgtacctg accttcttcctcgag gacgac 4381 AspValAspVal SerPhe MetTyrLeu ThrPhePheLeuGlu AspAsp gacaagctcgag cagatc aggaaggat tacaccagcggagcc atgctc 4429 AspLysLeuGlu GlnIle ArgLysAsp TyrThrSerGlyAla MetLeu accggtgagctc aagaag gcactcata gaggttctgcagccc ttgatc 4477 ThrGlyGluLeu LysLys AlaLeuIle GluVa1LeuGlnPro LeuIle gcagagcaccag gcccgg cgcaaggag gtcacggatgagata gtgaaa 4525 AlaGluHisGln AlaArg ArgLysGlu Val'?'hrAspGluIle ValLys gagttcatgact ccccgg aagctgtcc ttcgactttcagaag cttgcg 4573 GluPheMetThr ProArg LysLeuSer PheAspPheGlnLys LeuAla gccgcactcgag caccac caccaccac cactgagatccgg ctgctaacaa 4623 AlaAlaLeuGlu HisHis HisHisHis His agcccgaaag gaagctgagt tggctgctgc caccgctgag caataactag cataacccct 4683 tggggcctct aaacgggtct tgaggggttt tttgctgaaa ggaggaacta tatccggat 4742 <210> 16 <211> 392 <212> PRT
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: human inactive TrpRS in pET20B
<400> 16 Met Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg Pro His His Phe Leu Arg Arg Gly Ile Phe Phe Ser His Arg Asp Met Asn Gln Val Leu Asp Ala Tyr Glu Asn Lys Lys Pro Phe Tyr Leu Tyr Thr Gly Arg Gly Pro Ser Ser (~lu Ala Met His Val Gly His Leu Ile Pro Phe Ile Phe Thr Lys Trp Leu Gln Asp Val Phe Asn Val Pro Leu Val Ile Gln Met Thr Asp Asp Glu Lys Tyr Leu T:rp Lys Asp Leu Thr Leu Asp Gln Ala Tyr Gly Asp Ala Val Glu Asn ALa Lys Asp Ile Ile Ala Cys Gly Phe Asp Ile Asn Lys Thr Phe Ile Phe Ser Asp Leu Asp Tyr Met Gly Met Ser Ser Gly Phe Tyr Lys Asn Val Val Lys Ile Gln Lys His Val Thr Phe Asn Gln Val Lys Gly Ile Phe Gly 165 170 1'75 Phe Thr Asp Ser Asp Cys Ile Gly Lys Ile Ser Phe Pro Ala I1e G1n Ala Ala Pro Ser Phe Ser Asn Ser Phe Pro Gln Ile Phe Arg Asp Arg Thr Asp Ile Gln Cys Leu Ile Pro Cys Ala Ile Asp Gln Asp Pro Tyr Phe Arg Met Thr Arg Asp Val Ala Pro Arg Ile Gly Tyr Pro Lys Pro Ala Leu Leu His Ser Thr Phe Phe Pro Ala Leu Gln Gly Ala G1n Thr Lys Met Ser Ala Ser Asp Pro Asn Ser Ser Ile Phe Leu Thr Asp Thr Ala Lys Gln Ile Lys Thr Lys Val Asn Lys His Ala Phe Ser Gly Gly Arg Asp Thr Ile Glu Glu His Arg Gln Phe Gly Gly Asn Cys Asp Val Asp Val Ser Phe Met Tyr Leu Thr Phe Phe Leu Glu Asp Asp Asp Lys Leu Glu Gln Ile Arg Lys Asp Tyr Thr Ser Gly Ala Met Leu Thr Gly Glu Leu Lys Lys Ala Leu Ile Glu Val Leu Gln Pro Leu Ile Ala Glu His Gln Ala Arg Arg Lys Glu Val Thr Asp Giu Ile Val Lys Glu Phe Met Thr Pro Arg Lys Leu Ser Phe Asp Phe Gln Lys Leu Ala Ala Ala Leu Glu His His His His His His <210> 17 <211> 6 <212> PRT
<213> Homo sapiens <400> 17 Glu Leu Arg Val Ser Tyr <210> 18 <211> 6 <212> PRT
<213> Escherichia coli <400> 18 Glu Thr Val Gln Glu Trp <210> 19 <211> 9 <212> PRT
<213> Homo Sapiens <400> 19 Ser Ala Lys Glu Leu Arg Cys Gln Cys <210> 20 <211> 11 <212> PRT
<213> Homo Sapiens <400> 20 Ala Ser Val Ala Thr Glu Leu Arg Cys Gln Cys <210> 21 <211> 7 <212> PRT
<213> Homo Sapiens <400> 21 Ala Glu Leu Arg Cys Gln Cys <210> 22 <211> 58 <212> PRT

<213> Homo Sapiens <400> 22 Gly Asp Glu Lys Lys Ala Lys Glu Lys Ile Glu Lys Lys Gly G1u Lys Lys Glu Lys Lys G1n Gln Ser Ile Ala Gly ;per Ala Asp Ser Lys Pro Ile Asp Val Ser Arg Leu Asp Leu Arg I1e Gly Cys Ile Ile Thr Ala Arg Lys His Pro Asp Ala Asp Ser Leu Tyr <210> 23 <211> 58 <212> PRT
<213> Homo Sapiens <400> 23 Pro Ala Leu Lys Lys Leu Ala Ser Ala Ala Tyr Pro Asp Pro Ser Lys Gln Lys Pro Met Ala Lys Gly Pro Ala Lys Asn Ser Glu Pro G1u Glu Val Ile Pro Ser Arg Leu Asp Ile Arg Val Gly Lys Ile Ile Thr Val Glu Lys His Pro Asp Ala Asp Ser Leu Tyr <210> 24 <211> 7 <212> PRT
<213> Homo sapiens <400> 24 Arg Val Gly Lys Ile Ile Thr <210> 25 <211> 7 <212> PRT
<213> Homo Sapiens <400> 25 Arg Ile Gly Cys Ile Ile Thr <210> 26 <211> 7 <212> PRT
<213> Homo Sapiens <400> 26 Arg Ile Gly Arg Ile Ile Thr <210> 27 <211> 7 <212> PRT
<213> Caenorhabditis elegans <400> 27 Arg Val Gly Arg Ile Ile Lys <210> 28 <211> 7 <212> PRT
<213> Saccharomyces cerevisiae <400> 28 Arg Val Gly Phe Ile Gln Lys <210> 29 <211> 7 <212> PRT
<213> Bos taurus <400> 29 Arg Val Gly Lys Val Ile Ser <210> 30 <211> 7 <212> PRT
<213> Mus musculus <400> 30 Arg Ile Gly Cys Ile Val Thr <210> 31 <211> 7 <212> PRT
<213> Mesocricetus auratus <400> 31 Arg Ile Gly Arg Ile Val Thr <210> 32 <211> 7 <212> PRT

<213> Ovis aries <400> 32 Arg Ile Gly Cys Ile Ile Thr <210> 33 <211> 7 <212> PRT
<213> Calcarea sp.
<400> 33 Arg Ile Gly Arg Ile Thr Ser <210> 34 <211> 7 <212> PRT
<213> A. aeolicus <400> 34 Arg Val Ala Lys Val Leu Ser <210> 35 <211> 7 <212> PRT
<213> Escherichia coli <400> 35 Arg Val Gly Lys Ile Val Glu <210> 36 <211> 7 <212> PRT
<213> Escherichia coli <400> 36 Arg Val A1a Leu Ile Glu Asn <210> 37 <211> 7 <212> PRT
<213> Haemophilus influenzae <400> 37 Arg Val Ala Lys Val Leu Lys <210> 38 <211> 7 <212> PRT
<213> Bacillus subtilis <400> 38 Arg Val Ala Glu Val Ile Glu <210> 39 <211> 7 <212> PRT
<213> B. stearothermophilus <400> 39 Arg Val Ala Glu Val Val Gln <210> 40 <211> 7 <212> PRT
<213> Thermus thermophilus <400> 40 Arg Val Ala Glu Val Leu Ala <210> 41 <211> 6 <212> PRT
<213> Escherichia coli <400> 41 Val Gly Glu Val Val Glu <210> 42 <211> 6 <212> PRT
<213> Bacillus subtilis <400> 42 Ile Gly His Val Leu Glu <210> 43 <211> 6 <212> PRT
<213> Synechococcus sp.
<400> 43 Val Gly Arg Va1 Leu Glu <210> 44 <211> 6 <212> PRT
<213> Thermus thermophilus <400> 44 Phe Ala Arg Val Leu Glu <210> 45 <211> 85 <212> PRT
<213> Homo sapiens <400> 45 Met Ser Tyr Lys Ala Ala Ala Gly Glu Asp 'ryr Lys Ala Asp Cys Pro Pro G1y Asn Pro Ala Pro Thr Ser Asn His Gly Pro Asp Ala Thr Glu Ala Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr Ser Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Arg Phe Gly Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg Pro His His Phe Leu <210> 46 <211> 85 <212> PRT

<213> Bos taurus <400> 46 Thr Ser LysAlaAla ThrG.lyGlu AspTyrLysVal AspCys Pro Tyr Pro Gly ProAlaPro GluSerGly GluGlyL,euAsp AlaThr Glu Asp Ala Asp AspPheVal AspProTrp ThrValGlnThr SerSer Ala Glu Lys Gly AspTyrAsp LysLeuIle ValArgPheGly SerSer Lys Ile Ile Asp GluLeuVal AsnArgIle GluArgAlaThr GlyGln Arg Lys Pro His PheLeu Arg <210> 47 <211> 85 <212> PRT
<213> Mus musculus <400> 47 Met Ser Tyr Lys Ala Ala Met Gly Glu Glu 'ryr Lys Ala Gly Cys Pro Pro Gly Asn Pro Thr Ala Gly Arg Asn Cys Asp Ser Asp Ala Thr Lys Ala Ser Glu Asp Phe Val Asp Pro Trp Thr Val Arg Thr Ser Ser Ala Lys Gly Ile Asp Tyr Asp Lys Leu Ile Val Gln Pro Gly Ser Ser Lys Ile Asp Lys Glu Leu Ile Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg 65 70 ~5 80 Pro His Arg Phe Leu <210> 98 <211> 85 <212> PRT
<213> Oryctolagus cuniculus <400> 48 Thr Ser Tyr Lys Glu Ala Met Gly Glu Asp T yr Lys Ala Asp Cys Pro Pro Gly Asn Ser Thr Pro Asp Ser His Gly Pro Asp Glu Ala Val Asp Asp Lys G1u Asp Phe Val Asp Pro Trp Thr Val Arg Thr Ser Ser Ala Lys Gly I1e Asp Tyr Asp Lys Leu Ile Val Gln Phe Gly Ser Ser Lys Ile Asp Lys Glu Leu Val Asn Arg Ile Glu Arg Ala Thr Gly Gln Arg Pro His Arg Phe Leu <210> 49 <211> 86 <212> PRT
<213> Homo sapiens <400> 49 I1e Ser Tyr Gln Gly Arg Ile Pro Tyr Pro Arg Pro Gly Thr Cys Pro Gly Gly Ala Phe Thr Pro Asn Met Arg Thr T hr Lys Glu Phe Pro Asp Asp Val Val Thr Phe Ile Arg Asn His Pro Leu Met Tyr Asn Ser Ile Tyr Pro Ile His Lys Arg Pro Leu Ile Val Arg Ile Gly Thr Asp Tyr Lys Tyr Thr Lys Ile Ala Val Asp Arg Val Asn Ala Ala Asp Gly Arg Tyr His Val Leu Phe Leu <210> 50 <211> 86 <212> PRT
<213> Mus musculus <400> 50 Ile Ser Tyr Gln Gly Arg Ile Pro Tyr Pro Arg Pro Gly Thr Cys Pro Gly Gly Ala Phe Thr Pro Asn Met Arg Thr Thr hys Asp Phe Pro Asp Asp Val Val Thr Phe Ile Arg Asn His Pro Leu Met Tyr Asn Ser Ile Ser Pro Ile His Arg Arg Pro Leu Ile Val Arg Ile Gly Thr Asp Tyr Lys Tyr Thr Lys Ile Ala Val Asp Arg Val Asn Ala Ala Asp Gly Arg Tyr His Va1 Leu Phe Leu <210> 51 <211> 46 <212> PRT
<213> Homo Sapiens <400> 51 Ala Ala Ala Gly Glu Asp Tyr Lys Ala Asp Cys Pro Pro Gly Psn Pro Ala Pro Thr Ser Asn His Gly Pro Asp Ala Thr Glu Ala Glu Glu Asp Phe Val Asp Pro Trp Thr Val Gln Thr Ser Ser Ala Lys Gly <210> 52 <211> 46 <212> PRT
<213> Bos taurus <400> 52 Ala Ala Thr Gly Glu Asp Tyr Lys Val Asp Cys Pro Pro Gly Asp Pro Ala Pro Glu Ser Gly Glu Gly Leu Asp Ala 'rhr Glu Ala Asp Glu Asp Phe Val Asp Pro Trp Thr Val G1n Thr Ser Ser Ala Lys Gly <210> 53 <211> 46 <212> PRT
<213> Mus musculus <400> 53 Ala Ala Met Gly Glu Glu Tyr Lys Ala Gly Cys Pro Pro Gly Asn Pro Thr Ala Gly Arg Asn Cys Asp Ser Asp Ala Thr Lys Ala Ser Glu Asp Phe Val Asp Pro Trp Thr Val Arg Thr Ser Ser Ala Lys Gly <210> 54 <211> 46 <212> PRT

<213> Oryctolagus cuniculus <400> 54 Glu Ala GlyGlu Asp LysAla AspCys Pro Gly Asn Met Tyr Pro Ser Thr Pro SerHis Gly AspGlu AlaVal Asp Lys Glu Asp Pro Asp Asp Phe Val ProTrp Thr ArgThr SerSer Ala Gly Asp Val Lys <210> 55 <211> 41 <212> PRT
<213> Mus musculus <900> 55 Ala Phe Ala Gly Glu Asp Phe Lys Val Asp Ile Pro Glu Thr His Gly Gly Glu Gly Thr Glu Asp Glu Ile Asp Asp Glu 'ryr Glu Gly Asp Trp Ser Asn Ser Ser Ser Ser Thr Ser Gly <210> 56 <211> 5 <212> PRT
<213> Homo sapiens <400> 56 Met Gly Asp Ala Pro <210> 57 <211> 5 <212> PRT
<213> Homo Sapiens <400> 57 Ser Asn His Gly Pro <210> 58 <211> 5 <212> PRT
<213> Homo sapiens <400> 58 Ser Ala Lys Gly Ile

Claims

WHAT IS CLAIMED IS:

1. A composition of matter that comprises a library of analytes, said analytes being hybridized to an array of nucleic acids, said nucleic acids being fixed or immobilized to a solid support, wherein said analytes comprise an inherent universal detection target (UDT), and a universal detection element (UDE) attached to said UDT wherein said UDE generates a signal indicating the presence or quantity of said analytes, or said attachment of UDE to UDT.

2. The composition of claim 1, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

3. The composition of claim 1, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

4. The composition of claim 1, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

5. The composition of claim 4, wherein said analogs comprise PNA.

6. The composition of claims 4 or 5, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

7. The composition of claim 1, wherein said solid support is porous or non-porous.

8. The composition of claim 7, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

9. The composition of claim 7, wherein said non-porous solid support comprises glass or plastic.

10. The composition of claim 1, wherein said solid support is transparent, translucent, opaque or reflective.

11. The composition of claim 1, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

12. The composition of claim 11, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

13. The composition of claim 1, wherein said inherent UDT is selected from the group consisting of 3' polyA segments, 5' caps, secondary structures, consensus sequences and a combination of any of the foregoing.

14. The composition of claim 13, wherein said consensus sequences is selected from the group consisting of signal sequences for polyA addition, splicing elements, multicopy repeats and a combination of any of the foregoing.

15. The composition of claim 1, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, synthetic polymers and a combination of any of the foregoing.

16. The composition of claim 4, wherein said analogs comprise PNA.

17. The composition of claim 1, wherein said UDE generates a signal directly or indirectly.

18. The composition of claim 17, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

19. The composition of claim 17, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

20. The composition of claim 19, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

21. A composition of matter that comprises a library of analytes, said analytes being hybridized to an array of nucleic acids, said nucleic acids being fixed or immobilized to a solid support, wherein said analytes comprise a non-inherent universal detection target (UDT) and a universal detection element (UDE) hybridized to said UDT, wherein said UDE generates a signal directly or indirectly to detect the presence or quantity of said analytes.

22. The composition of claim 21, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

23. The composition of claim 21, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

24. The composition of claim 21, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

25. The composition of claim 24, wherein said analogs comprise PNA.

26. The composition of claims 24 or 25, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

27. The composition of claim 21, wherein said solid support is porous or non-porous.

28. The composition of claim 27, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

29. The composition of claim 27, wherein said non-porous solid support comprises glass or plastic.

30. The composition of claim 21, wherein said solid support is transparent, translucent, opaque or reflective.

31. The composition of claim 21, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

32. The composition of claim 31, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

33. The composition of claim 21, wherein said non-inherent universal detection target (UDT) comprises homopolymeric sequences.

34. The composition of claim of 21, wherein said non-inherent universal detection target (UDT) comprises heteropolymeric sequences.

35. The composition of claim 21, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs and modified forms thereof.

36. The composition of claim 35, wherein said analogs comprise PNA.

37. The composition of claim 21, wherein said UDE generates a signal directly or indirectly.

38. The composition of claim 37, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

39. The composition of claim 37, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

40. The composition of claim 39, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

41. A composition of matter that comprises a library of analytes, said analytes being hybridized to an array of nucleic acids, said nucleic acids being fixed or immobilized to a solid support, wherein said hybridization between said analytes and said nucleic acids generate a domain for complex formation, and said composition further comprising a signaling entity complexed to said domain.

42. The composition of claim 41, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

43. The composition of claim 41, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

44. The composition of claim 41, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

45. The composition of claim 44, wherein said analogs comprise PNA.

46. The composition of claims 44 or 45, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

47. The composition of claim 41, wherein said solid support is porous or non-porous.

48. The composition of claim 47, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

49. The composition of claim 47, wherein said non-porous solid support comprises glass or plastic.

50. The composition of claim 41, wherein said solid support is transparent, translucent, opaque or reflective.

51. The composition of claim 41, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

52. The composition of claim 41, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

53. The composition of claim 41, wherein said domain for complex formation is selected from the group consisting of DNA-DNA hybrids, DNA-RNA hybrids, RNA-RNA hybrids, DNA-PNA hybrids and RNA-PNA hybrids.

54. The composition of claim 41, wherein said signaling entity complexed to said domain is selected from the group consisting of proteins and intercalators.

55. The composition of claim 54, wherein said proteins comprise nucleic acid binding proteins which bind preferentially to double-stranded nucleic acid.

56. The composition of claim 55, wherein said nucleic acid binding proteins comprise antibodies.

57. The composition of claim 56, wherein said antibodies are specific for nucleic acid hybrids selected from the group consisting of DNA-DNA hybrids, DNA-RNA
hybrids, RNA-RNA hybrids, DNA-PNA hybrids and RNA-PNA hybrids

58. The composition of claim 54, wherein said intercalators are selected from the group consisting of ethidium bromide, diethidium bromide, acridine orange and SYBR Green.

59. The composition of claim 41, wherein said proteins generate a signal directly or indirectly.

60. The composition of claim 59, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

61. The composition of claim 59, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

62. The composition of claim 61, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

63. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of said nucleic acids of interest comprise at least one inherent universal detection target (UDT); and (iii) universal detection elements (UDE) which generates a signal directly or indirectly;
b) hybridizing said library (ii) with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present;

c) contacting said UDEs with said UDTs to form a complex bound to said array;
d) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

64. The process of claim 63, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

65. The process of claim 64, wherein said analogs comprise PNA.

66. The process of claims 64 or 65, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

67. The process of claim 63, wherein said solid support is porous or non-porous.

68. The process of claim 67, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

69. The process of claim 67, wherein said non-porous solid support comprises glass or plastic.

70. The process of claim 63, wherein said solid support is transparent, translucent, opaque or reflective.

71. The process of claim 63, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

72. The process of claim 71, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

73. The process of claim 63, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

74. The process of claim 63, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

75. The process of claim 63, wherein said inherent UDT is selected from the group consisting of 3' polyA segments, 5' caps, secondary structures, consensus sequences, and a combination of any of the foregoing.

76. The process of claim 75, wherein said consensus sequences is selected from the group consisting of signal sequences for polyA addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

77. The process of claim 63, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, synthetic polymers and a combination of any of the foregoing.

78. The process of claim 64, wherein said analogs comprise PNA.

79. The process of claim 63, wherein said UDE generates a signal directly or indirectly.

80. The process of claim 79, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

81. The process of claim 79, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

82. The process of claim 81, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

83. The process of claim 63, comprising one or more washing steps.

84. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of said nucleic acids of interest comprise at least one inherent universal detection target (UDT); and (iii) universal detection elements (UDE) which generates a signal directly or indirectly;
b) contacting said UDEs with said UDTs in said library of nucleic acid analytes to form one or more complexes;
c) hybridizing said library of nucleic acid analytes with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present;
d) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

85. The process of claim 84, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

86. The process of claim 85, wherein said analogs comprise PNA.

87. The process of claims 85 or 86, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

88. The process of claim 84, wherein said solid support is porous or non-porous.

89. The process of claim 88, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

90. The process of claim 88, wherein said non-porous solid support comprises glass or plastic.

91. The process of claim 84, wherein said solid support is transparent, translucent, opaque or reflective.

92. The process of claim 84, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

93. The process of claim 92, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

94. The process of claim 84, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

95. The process of claim 84, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

96. The process of claim 84, wherein said inherent UDT is selected from the group consisting of 3' polyA segments, 5' caps, secondary structures, consensus sequences, and a combination of any of the foregoing.

97. The process of claim 96, wherein said consensus sequences is selected from the group consisting of signal sequences for polyA addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

98. The process of claim 84, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, synthetic polymers and a combination of any of the foregoing.

99. The process of claim 98, wherein said analogs comprise PNA.

100. The process of claim 84, wherein said UDE generates a signal directly or indirectly.

101. The process of claim 100, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

102. The process of claim 100, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

103. The process of claim 102, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

104. The process of claim 84, comprising one or more washing steps.

105. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of said nucleic acids of interest comprise at least one non-inherent universal detection target (UDT), wherein said non-inherent UDT is attached to said nucleic acid analytes; and (iii) universal detection elements (UDE) which generate a signal directly or indirectly;
b) hybridizing said library (ii) with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present;
c) contacting said UDEs with said UDTs to form a complex bound to said array;
d) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

106. The process of claim 105, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

107. The process of claim 106, wherein said analogs comprise PNA.

108. The process of claims 106 or 107, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

109. The process of claim 105, wherein said solid support is porous or non-porous.

110. The process of claim 109, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

111. The process of claim 109, wherein said non-porous solid support comprises glass or plastic.

112. The process of claim 105, wherein said solid support is transparent, translucent, opaque or reflective.

113. The process of claim 105, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

114. The process of claim 113, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

115. The process of claim 105, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

116. The process of claim 105, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

117. The process of claim 105, wherein said non-inherent universal detection target (UDT) comprises homopolymeric sequences.

118. The process of claim of 105, wherein said non-inherent universal detection target (UDT) comprises heteropolymeric sequences.

119. The process of claim 105, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs and modified forms thereof.

120. The process of claim 119, wherein said analogs comprise PNA.

121. The process of claim 105, wherein said UDE generates a signal directly or indirectly.

122. The process of claim 121, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

123. The process of claim 121, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

124. The process of claim 123, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

125. The process of claim 105, comprising one or more washing steps.

126. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of said nucleic acids of interest comprise at least one non-inherent universal detection target (UDT), wherein said non-inherent UDTs are attached to said nucleic acid analytes; and (iii) universal detection elements (UDE) which generate a signal directly or indirectly;
b) contacting said UDEs with said UDTs in said library of nucleic acid analytes to form one or more complexes;
c) hybridizing said library (ii) with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present;

d) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

127. The process of claim 126, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

128. The process of claim 127, wherein said analogs comprise PNA.

129. The process of claims 127 or 128, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

130. The process of claim 126, wherein said solid support is porous or non-porous.

131. The process of claim 130, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

132. The process of claim 130, wherein said non-porous solid support comprises glass or plastic.

133. The process of claim 126, wherein said solid support is transparent, translucent, opaque or reflective.

134. The process of claim 126, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

135. The process of claim 134, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

136. The process of claim 126, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

137. The process of claim 126, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

138. The process of claim 126, wherein said non-inherent universal detection target (UDT) comprises homopolymeric sequences.

139. The process of claim of 126, wherein said non-inherent universal detection target (UDT) comprises heteropolymeric sequences.

140. The process of claim 126, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs and modified forms thereof.

141. The process of claim 140, wherein said analogs comprise PNA.

142. The process of claim 126, wherein said UDE generates a signal directly or indirectly.

143. The process of claim 142, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

144. The process of claim 142, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

145. The process of claim 144, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

146. The process of claim 126, comprising one or more washing steps.

147. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more universal detection targets (UDT) to a nucleic acid;

(iv) universal detection elements (UDE) which generates a signal directly or indirectly;
b) attaching said UDTs (iii) to said library of nucleic acid analytes (ii);
c) hybridizing said library (ii) with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present;
d) contacting said UDEs with said UDTs to form a complex bound to said array;
e) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

148. The process of claim 147, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

149. The process of claim 148, wherein said analogs comprise PNA.

150. The process of claims 148 or 149, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

151. The process of claim 147, wherein said solid support is porous or non-porous.

152. The process of claim 151, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

153. The process of claim 151, wherein said non-porous solid support comprises glass or plastic.

154. The process of claim 147, wherein said solid support is transparent, translucent, opaque or reflective.

155. The process of claim 147, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

156. The process of claim 155, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

157. The process of claim 147, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

158. The process of claim 147, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

159. The process of claim 147, wherein said attaching means add homopolymeric sequences through an enzyme selected from the group consisting of poly A
polymerase and terminal transferase.

160. The process of claim 147, wherein said attaching means add homopolymeric or heteropolymeric sequences through an enzyme selected from the group consisting of DNA ligase and RNA ligase.

161. The process of claim 147, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs and modified forms thereof.

162. The process of claim 161, wherein said analogs comprise PNA.

163. The process of claim 147, wherein said UDE generates a signal directly or indirectly.

164. The process of claim 163, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

165. The process of claim 163, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

166. The process of claim 165, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

167. The process of claim 147, comprising one or more washing steps.

168. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more universal detection targets (UDT) to a nucleic acid;
(iv) universal detection elements (UDE) which generates a signal directly or indirectly;
b) attaching said UDTs (iii) to said library of nucleic acid analytes (ii);
c) contacting said UDEs with said UDTs in said library of nucleic acid analytes to form one or more complexes;
d) hybridizing said library (ii) with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present;
e) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

169. The process of claim 168, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

170. The process of claim 169, wherein said analogs comprise PNA.

171. The process of claims 169 or 170, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

172. The process of claim 168, wherein said solid support is porous or non-porous.

173. The process of claim 172, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

174. The process of claim 172, wherein said non-porous solid support comprises glass or plastic.

175. The process of claim 168, wherein said solid support is transparent, translucent, opaque or reflective.

176. The process of claim 168, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

177. The process of claim 176, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

178. The process of claim 168, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

179. The process of claim 168, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

180. The process of claim 168, wherein said attaching means add homopolymeric sequences through an enzyme selected from the group consisting of poly A
polymerase and terminal transferase.

181. The process of claim 168, wherein said attaching means add homopolymeric or heteropolymeric sequences through an enzyme selected from the group consisting of DNA ligase and RNA ligase.

182. The process of claim 168, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs and modified forms thereof.

183. The process of claim 182, wherein said analogs comprise PNA.

184. The process of claim 168, wherein said UDE generates a signal directly or indirectly.

185. The process of claim 184, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

186. The process of claim 184, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

187. The process of claim 186, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

188. The process of claim 168, comprising one or more washing steps.

189. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) universal detection elements (UDEs) which bind to a domain formed by nucleic acid hybrids for complex formation and generate a signal directly or indirectly;
b) hybridizing said library (ii) with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present, wherein said formed hybrids generate a domain for complex formation;

c) contacting said UDEs with said hybrids to form a complex bound to said array;
d) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

190. The process of claim 189, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

191. The process of claim 190, wherein said analogs comprise PNA.

192. The process of claims 190 or 191, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

193. The process of claim 189, wherein said solid support is porous or non-porous.

194. The process of claim 193, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

195. The process of claim 193, wherein said non-porous solid support comprises glass or plastic.

196. The process of claim 189, wherein said solid support is transparent, translucent, opaque or reflective.

197. The process of claim 189, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

198. The process of claim 197, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

199. The process of claim 189, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

200. The process of claim 189, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

201. The process of claim 189, wherein said domain for complex formation is selected from the group consisting of DNA-DNA hybrids, DNA-RNA hybrids, RNA-RNA hybrids, DNA-PNA hybrids and RNA-PNA hybrids.

202. The process of claim 189, wherein said signaling entity complexed to said domain is selected from the group consisting of proteins and intercalators.

203. The process of claim 202, wherein said proteins comprise nucleic acid binding proteins which bind preferentially to double-stranded nucleic acid.

204. The process of claim 203, wherein said nucleic acid binding proteins comprise antibodies.

205. The process of claim 204, wherein said antibodies are specific for nucleic acid hybrids selected from the group consisting of DNA-DNA hybrids, DNA-RNA
hybrids, RNA-RNA hybrids, DNA-PNA hybrids and RNA-PNA hybrids.

206. The process of claim 202, wherein said intercalators are selected from the group consisting of ethidium bromide, diethidium bromide, acridine orange and SYBR Green.

207. The process of claim 189, wherein said proteins generate a signal directly or indirectly.

208. The process of claim 207, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

209. The process of claim 207, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

210. The process of claim 209, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

211. The process of claim 189, further comprising one or more washing steps.

Paga 183

212. A composition of matter comprising a library of first nucleic acid analyte copies, said first nucleic acid copies being hybridized to an array of nucleic acids, said nucleic acids being fixed or immobilized to a solid support, wherein said first nucleic acid copies comprise an inherent universal detection target (UDT) and a universal detection element (UDE) attached to said UDT, wherein said UDE
generates a signal directly or indirectly to detect the presence or quantity of said analytes.

213. The composition of claim 212, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

214. The composition of claim 212, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

215. The composition of claim 212, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

216. The composition of claim 215, wherein said analogs comprise PNA.

217. The composition of claims 215 or 216, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

218. The composition of claim 212, wherein said solid support is porous or non-porous.

219. The composition of claim 218, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

220. The composition of claim 218, wherein said non-porous solid support comprises glass or plastic.

221. The composition of claim 212, wherein said solid support is transparent, translucent, opaque or reflective.

222. The composition of claim 212, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

223. The composition of claim 222, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

224. The composition of claim 212, wherein said inherent UDT is selected from the group consisting of poly T segments, secondary structures, consensus sequences, and a combination of any of the foregoing.

225. The composition of claim 224, wherein said consensus sequences is selected from the group consisting of signal sequences for polyA addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

226. The composition of claim 212, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, synthetic polymers and a combination of any of the foregoing.

227. The composition of claim 226, wherein said analogs comprise PNA.

228. The composition of claim 212, wherein said UDE generates a signal directly or indirectly.

229. The composition of claim 228, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

230. The composition of claim 228, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

231. The composition of claim 230, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

232. A composition of matter comprising a library of first nucleic acid copies, said first nucleic acid copies being hybridized to an array of nucleic acids, said nucleic acids being fixed or immobilized to a solid support, wherein said first nucleic acid copies comprise one or more non-inherent universal detection targets (UDTs) and one or more universal detection elements (UDEs) attached to said UDTs, wherein said UDEs generate a signal directly or indirectly to detect the presence or quantity of said analytes, and wherein said UDTs are either: (i) at the 5' ends of said first nucleic acid copies and not adjacent to an oligoT segment or sequence, or (ii) at the 3' ends of said first nucleic acid copies, or (iii) both (i) and (ii).

233. The composition of claim 232, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

234. The composition of claim 232, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

235. The composition of claim 232, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

236. The composition of claim 235, wherein said analogs comprise PNA.

237. The composition of claims 235 or 236, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

238. The composition of claim 232, wherein said solid support is porous or non-porous.

239. The composition of claim 238, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

240. The composition of claim 238, wherein said non-porous solid support comprises glass or plastic.

241. The composition of claim 232, wherein said solid support is transparent, translucent, opaque or reflective.

242. The composition of claim 232, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

243. The composition of claim 242, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

244. The composition of claim 232, wherein said non-inherent universal detection target (UDT) comprises homopolymeric sequences.

245. The composition of claim of 232, wherein said non-inherent universal detection target (UDT) comprises heteropolymeric sequences.

246. The composition of claim 232, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, synthetic polymers and a combination of any of the foregoing.

247. The composition of claim 246, wherein said analogs comprise PNA.

248. The composition of claim 232, wherein said UDE generates a signal directly or indirectly.

249. The composition of claim 248, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

250. The composition of claim 248, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

251. The composition of claim 250, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

252. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:

a) providing:
(i) an array of fixed or immobilized nucleic acids identical in part or whole to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of said nucleic acids of interest comprise at least one inherent universal detection target (UDT);
(iii) universal detection elements (UDE) which generate a signal directly or indirectly; and (iv) polymerizing means for synthesizing nucleic acid copies of said nucleic acids of analytes;

b) synthesizing one or more first nucleic acid copies which are complementary to all or part of said nucleic acid analytes and synthesizing sequences which are complementary to all or part of said UDT to form a complementary UDT;

c) hybridizing said first nucleic acid copies with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present;

d) contacting said UDEs with said complementary UDTs of said first nucleic acid copies to form a complex bound to said array;

e) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

253. The process of claim 252, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

254. The process of claim 253, wherein said analogs comprise PNA.

255. The process of claims 253 or 254, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

256. The process of claim 252, wherein said solid support is porous. or non-porous.

257. The process of claim 256, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

258. The process of claim 256, wherein said non-porous solid support comprises glass or plastic.

259. The process of claim 252, wherein said solid support is transparent, translucent, opaque or reflective.

260. The process of claim 252, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

261. The process of claim 260, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

262. The process of claim 252, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

263. The process of claim 252, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

264. The process of claim 252, wherein said inherent UDT is selected from the group consisting of poly T segments, secondary structures, consensus sequences, and a combination of any of the foregoing.

265. The process of claim 264, wherein said consensus sequences is selected from the group consisting of signal sequences for polyA addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

266. The process of claim 252, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, synthetic polymers and a combination of any of the foregoing.

267. The process of claim 266, wherein said analogs comprise PNA.

268. The process of claim 252, wherein said UDE generates a signal directly or indirectly.

269. The process of claim 268, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

270. The process of claim 268, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

271. The process of claim 270, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

272. The process of claim 252, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polyrnerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

273. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) ~providing:
(i) an array of fixed or immobilized nucleic acids identical in part or whole to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified, wherein each of said nucleic acids of interest comprise at least one inherent universal detection target (UDT);
(iii) universal detection elements (UDE) which generate a signal directly or indirectly; and (iv) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes;
b) synthesizing one or more first nucleic acid copies of said nucleic acid analytes;
c) contacting said UDEs with said UDTs in said first nucleic acid copies to form one or more complexes;
d) hybridizing said first nucleic acid copies with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present; and e) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

274. The process of claim 273, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

275. The process of claim 274, wherein said analogs comprise PNA.

276. The process of claims 274 or 275, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

277. The process of claim 273, wherein said solid support is porous or non-porous.

278. The process of claim 277, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

279. The process of claim 277, wherein said non-porous solid support comprises glass or plastic.

280. The process of claim 273, wherein said solid support is transparent, translucent, opaque or reflective.

281. The process of claim 273, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

282. The process of claim 281, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

283. The process of claim 273, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

284. The process of claim 273, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

285. The process of claim 273, wherein said inherent UDT is selected from the group consisting of poly T segments, secondary structures, consensus sequences, and a combination of any of the foregoing.

286. The process of claim 285, wherein said consensus sequences is selected from the group consisting of signal sequences for polyA addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

287. The process of claim 273, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs, polypeptides, polysaccharides, synthetic polymers and a combination of any of the foregoing.

288. The process of claim 287, wherein said analogs comprise PNA.

289. The process of claim 273, wherein said UDE generates a signal directly or indirectly.

290. The process of claim 289, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

291. The process of claim 289, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

292. The process of claim 291, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

293. The process of claim 283, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

294. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical in part or whole to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid;
(iv) universal detection elements (UDE) which generate a signal directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes;

b) attaching said non-inherent UDTs to either the 3' ends of said nucleic acid analytes, the 5' ends of said first nucleic acid analytes, or both said 3' ends and said 5' ends of said nucleic acid analytes;
c) synthesizing one or more first nucleic acid copies of said nucleic acid analytes;
d) hybridizing said first nucleic acid copies with said array of nucleic acids (I) to form hybrids if said nucleic acids of interest are present;
e) contacting said UDEs with said UDTs of said first nucleic acid copies to form a complex bound to said array; and f) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

295. The process of claim 294, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

296. The process of claim 295, wherein said analogs comprise PNA.

297. The process of claims 295 or 296, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

298. The process of claim 294, wherein said solid support is porous or non-porous.

299. The process of claim 298, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

300. The process of claim 298, wherein said non-porous solid support comprises glass or plastic.

301. The process of claim 294, wherein said solid support is transparent, translucent, opaque or reflective.

302. The process of claim 294, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

303. The process of claim 302, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

304. The process of claim 294, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

305. The process of claim 294, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

306. The process of claim 294, wherein said attaching means add homopolymeric sequences through an enzyme selected from the group consisting of poly A
polymerase and terminal transferase.

307. The process of claim 294, wherein said attaching means add homopolymeric or heteropolymeric sequences through an enzyme selected from the group consisting of DNA ligase and RNA ligase.

308. The process of claim 294, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs and modified forms thereof.

309. The process of claim 308, wherein said analogs comprise PNA.

310. The process of claim 294, wherein said UDE generates a signal directly or indirectly.

311. The process of claim 310, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

312. The process of claim 310, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

313. The process of claim 312, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

314. The process of claim 294, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

315. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical in part or whole to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid;
(iv) universal detection elements (UDE) which generate a signal directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes;
b) attaching said non-inherent UDTs to either the 3' ends of said nucleic acid analytes, the 5' ends of said first nucleic acid analytes, or both said 3' ends and said 5' ends of said nucleic acid analytes;
c) synthesizing one or more first nucleic acid copies of said nucleic acid analytes;

d) contacting said UDEs with said UDTs of said first nucleic acid copies to form complexes;
e) hybridizing said first nucleic acid copies with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present;
f) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

316. The process of claim 315, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

317. The process of claim 316, wherein said analogs comprise PNA.

318. The process of claims 316 or 317, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

319. The process of claim 315, wherein said solid support is porous or non-porous.

320. The process of claim 319, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

321. The process of claim 319, wherein said non-porous solid support comprises glass or plastic.

322. The process of claim 315, wherein said solid support is transparent, translucent, opaque or reflective.

323. The process of claim 315, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

324. The process of claim 323, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

325. The process of claim 315, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

326. The process of claim 315, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

327. The process of claim 315, wherein said attaching means add homopolymeric sequences through an enzyme selected from the group consisting of poly A
polymerase and terminal transferase.

328. The process of claim 315, wherein said attaching means add homopolymeric or heteropolymeric sequences through an enzyme selected from the group consisting of DNA ligase and RNA ligase.

329. The process of claim 315, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs and modified forms thereof.

330. The process of claim 329, wherein said analogs comprise PNA.

331. The process of claim 315, wherein said UDE generates a signal directly or indirectly.

332. The process of claim 331, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

333. The process of claim 331, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

334. The process of claim 333, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

335. The process of claim 315, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

336. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical in part or whole to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid;
(iv) universal detection elements (UDE) which generate a signal directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes;
b) synthesizing one or more first nucleic acid copies of said nucleic acid analytes;
c) attaching said non-inherent UDTs to either the 3' ends of said first nucleic acid copies, the 5' ends of said first nucleic acid copies, or both said 3' ends and said 5' ends of said first nucleic acid copies;
d) hybridizing said first nucleic acid copies with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present;
e) contacting said UDEs with said UDTs of said first nucleic acid copies to form a complex bound to said array;

f) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

337. The process of claim 336, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

338. The process of claim 337, wherein said analogs comprise PNA.

339. The process of claims 337 or 338, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

340. The process of claim 336, wherein said solid support is porous or non-porous.

341. The process of claim 340, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

342. The process of claim 340, wherein said non-porous solid support comprises glass or plastic.

343. The process of claim 336, wherein said solid support is transparent, translucent, opaque or reflective.

344. The process of claim 336, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

345. The process of claim 344, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

346. The process of claim 336, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

347. The process of claim 336, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

348. The process of claim 336, wherein said attaching means add homopolymeric sequences through terminal transferase.

349. The process of claim 336, wherein said attaching means add homopolymeric or heteropolymeric sequences through an enzyme selected from the group consisting of DNA ligase and RNA ligase.

350. The process of claim 336, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs and modified forms thereof.

351. The process of claim 350, wherein said analogs comprise PNA.

352. The process of claim 336, wherein said UDE generates a signal directly or indirectly.

353. The process of claim 352, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

354. The process of claim 352, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

355. The process of claim 354, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

356. The process of claim 336, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

357. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical in part or whole to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;

(iii) means for attaching one or more non-inherent universal detection targets (UDT) to a nucleic acid;
(iv) universal detection elements (UDE) which generate a signal directly or indirectly; and (v) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes;
b) synthesizing one or more first nucleic acid copies of said nucleic acid analytes;
c) attaching said non-inherent UDTs to either the 3' ends of said first nucleic acid copies, the 5' ends of said first nucleic acid copies, or both said 3' ends and said 5' ends of said first nucleic acid copies;
d) contacting said UDEs with said UDTs of said first nucleic acid copies to form a complex;
e) hybridizing said first nucleic acid copies with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present; and f) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

358. The process of claim 357, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

359. The process of claim 358, wherein said analogs comprise PNA.

360. The process of claims 358 or 359, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

361. The process of claim 357, wherein said solid support is porous or non-porous.

362. The process of claim 361, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

363. The process of claim 361, wherein said non-porous solid support comprises glass or plastic.

364. The process of claim 357, wherein said solid support is transparent, translucent, opaque or reflective.

365. The process of claim 357, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

366. The process of claim 365, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

367. The process of claim 357, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

368. The process of claim 357, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

369. The process of claim 357, wherein said attaching means add homopolymeric sequences through terminal transferase.

370. The process of claim 357, wherein said attaching means add homopolymeric or heteropolymeric sequences through an enzyme selected from the group consisting of DNA ligase and RNA ligase.

371. The process of claim 357, wherein said UDE is selected from the group consisting of nucleic acids, nucleic acid analogs and modified forms thereof.

372. The process of claim 371, wherein said analogs comprise PNA.

373. The process of claim 357, wherein said UDE generates a signal directly or indirectly.

374. The process of claim 373, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

375. The process of claim 373, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

376. The process of claim 375, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

377. The process of claim 357, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

378. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids complementary to said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) universal detection elements (UDEs) which bind to a domain for complex formation formed by nucleic acid hybrids and generate a signal directly or indirectly; and (iv) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes;
b) synthesizing one or more nucleic acid copies of said nucleic acid analytes;

c) hybridizing said first nucleic acid copies with said array of nucleic acids (i) to form hybrids if said nucleic acids of interest are present, wherein said formed hybrids generate a domain for complex formation;
d) contacting said UDEs with said hybrids to form a complex bound to said array; and e) detecting or quantifying said more than one nucleic acid of interest by detecting or measuring the amount of signal generated from UDEs bound to said array.

379. The process of claim 378, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

380. The process of claim 379, wherein said analogs comprise PNA.

381. The process of claims 379 or 380, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

382. The process of claim 378, wherein said solid support is porous or non-porous.

383. The process of claim 382, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

384. The process of claim 382, wherein said non-porous solid support comprises glass or plastic.

385. The process of claim 378, wherein said solid support is transparent, translucent, opaque or reflective.

386. The process of claim 378, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

387. The process of claim 386, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

388. The process of claim 378, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

389. The process of claim 378, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

390. The process of claim 378, wherein said domain for complex formation is selected from the group consisting of DNA-DNA hybrids, DNA-RNA hybrids, RNA-RNA hybrids, DNA-PNA hybrids and RNA-PNA hybrids.

391. The process of claim 378, wherein said signaling entity complexed to said domain is selected from the group consisting of proteins and intercalators.

392. The process of claim 391, wherein said proteins comprise nucleic acid binding proteins which bind preferentially to double-stranded nucleic acid.

393. The process of claim 392, wherein said nucleic acid binding proteins comprise antibodies.

394. The process of claim 393, wherein said antibodies are specific for nucleic acid hybrids selected from the group consisting of DNA-DNA hybrids, DNA-RNA
hybrids, RNA-RNA hybrids, DNA-PNA hybrids and RNA-PNA hybrids

395. The process of claim 391, wherein said intercalators are selected from the group consisting of ethidium bromide, diethidium bromide, acridine orange and SYBR Green.

396. The process of claim 391, wherein said protein generates a signal directly or indirectly.

397. The process of claim 396, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

398. The process of claim 396, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

399. The process of claim 398, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

400. The process of claim 378, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

401. A composition of matter comprising a library of double-stranded nucleic acids substantially incapable of in vivo replication and free of non-inherent homopolymeric sequences, said nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample, wherein said double-stranded nucleic acids comprise at least one inherent universal detection target (UDT) proximate to one end of said double strand and at least one non-inherent production center proximate to the other end of said double strand.

402. The composition of claim 401, wherein said sample comprises a biological source selected from the group consisting of organs, tissues and cells.

403. The composition of claim 401, wherein said library of nucleic acids are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

404. The composition of claim 401, wherein said inherent UDT is selected from the group consisting of 3' polyA segments, consensus sequences, or both.

405. The composition of claim 404, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

406. The composition of claim 401, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

407. The composition of claim 406, wherein said RNA promoters comprise phage promoters.

408. The composition of claim 407, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

409. A composition of matter comprising a library of double-stranded nucleic acids substantially incapable of in vivo replication, said nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample, wherein said double-stranded nucleic acids comprise at least four (4) non-inherent nucleotides proximate to one end of said double strand and a non-inherent production center proximate to the other end of said double strand.

410. The composition of claim 409, wherein said sample comprises a biological source selected from the group consisting of organs, tissues and cells.

411. The composition of claim 409, wherein said library of nucleic acids are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

412. The composition of claim 409, further comprising one or more inherent UDTs selected from the group consisting of 3' polyA segments, consensus sequences, or both.

413. The composition of claim 412, wherein said consensus sequences is selected from the group consisting of signal sequences for polyA addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

414. The composition of claim 409, wherein said at least four (4) non-inherent nucleotides are homopolymeric.

415. The composition of claim 409, wherein said non-inherent production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

416. The composition of claim 415, wherein said RNA promoters comprise phage promoters.

417. The composition of claim 416, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

418. A composition of matter comprising a library of double-stranded nucleic acids fixed to a solid support, said nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample and said nucleic acids further comprising at least one first sequence segment of non-inherent nucleotides proximate to one end of said double strand and at least one second sequence segment proximate to the other end of said double strand, said second sequence segment comprising at least one production center.

419. The composition of claim 418, wherein said solid support comprises beads.

420. The composition of claim 419, wherein said beads are magnetic.

421. The composition of claim 418, wherein said sample comprises a biological source selected from the group consisting of organs, tissues and cells.

422. The composition of claim 418, wherein said library of nucleic acids are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

423. The composition of claim 418, further comprising one or more inherent UDTs selected from the group consisting of 3' poly A segments, consensus sequences, or both.

424. The composition of claim 423, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

425. The composition of claim 418, wherein said non-inherent production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

426. The composition of claim 425, wherein said RNA promoters comprise phage promoters.

427. The composition of claim 426, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

428. A composition of matter comprising a library of double-stranded nucleic acids attached to a solid support, said nucleic acids comprising sequences complementary or identical in part or whole to inherent sequences of a library obtained from a sample, wherein said double-stranded nucleic acids comprise at least one inherent universal detection target (UDT) proximate to one end of said double strand and at least one non-inherent production center proximate to the other end of said double strand.

429. The composition of claim 428, wherein said solid support comprises beads.

430. The composition of claim 429, wherein said beads are magnetic.

431. The composition of claim 428, wherein said sample comprises a biological source selected from the group consisting of organs, tissues and cells.

432. The composition of claim 428, wherein said library of nucleic acids are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

433. The composition of claim 428, wherein said inherent UDT is selected from the group consisting of 3' polyA segments, consensus sequences, or both.

434. The composition of claim 433, wherein said consensus sequences is selected from the group consisting of signal sequences for polyA addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

435. The composition of claim 428, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

436. The composition of claim 435, wherein said RNA promoters comprise phage promoters.

437. The composition of claim 436, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

438. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers and a second set of primers, wherein said second set of primers comprises at least two segments, the first segment at the 3' end comprising random sequences, and the second segment comprising at least one production center;
(iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions;
b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity;
c) extending said bound first set of primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;
d) contacting said extended first copies with said second set of primers to form more than one second bound entity;
e) extending said bound second set of primers by means of template sequences provided by said extended first copies to form more than one complex comprising extended first copies and extended second set of primers;
f) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;
g) hybridizing said nucleic acid copies formed in step f) to said array of nucleic acids provided in step a) (i); and h) detecting or quantifying any of said hybridized copies obtained in step g).

439. The process of claim 438, wherein said nucleic acid array comprises members selected from the group consisting of DNA, RNA and analogs thereof.

440. The process of claim 439, wherein said analogs comprise PNA.

441. The process of claims 439 or 440, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

442. The process of claim 438, wherein said nucleic acid array is fixed or immobilized to a solid support.

443. The process of claim 442, wherein said solid support is porous or non-porous.

444. The process of claim 443, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

445. The process of claim 443, wherein said non-porous solid support comprises glass or plastic.

446. The process of claim 442, wherein said solid support is transparent, translucent, opaque or reflective.

447. The process of claim 442, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

448. The process of claim 447, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

449. The process of claim 438, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

450. The process of claim 438, wherein said library of nucleic acids analytes are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

451. The process of claim 438, wherein said first set of primers are complementary to inherent UDTs.

452. The process of claim 438, wherein said inherent UDT is selected from the group consisting of 3' poly A segments, consensus sequences, and a combination of both.

453. The process of claim 452, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

454. The process of claim 438, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

455. The process of claim 454, wherein said RNA promoters comprise phage promoters.

456. The process of claim 455, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

457. The process of claim 438, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

458. The process of claim 457, wherein said signaling entities generate a signal directly or indirectly.

459. The process of claim 458, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

460. The process of claim 458, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

461. The process of claim 460, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

462. The process of claim 438, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

463. The process of claim 438, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

464. The process of claim 438, wherein step g) is carried out repeatedly.

465. The process of claim 438, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1. reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

466. The process of claim 438, wherein said, means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

467. A process far detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers and a second set of primers, wherein said first set of primers comprise at least one production center; and (iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions;
b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity;
c) extending said bound first set of primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;
d) extending said first copies by means of at least four (4) or more non-inherent homopolymeric nucleotides;
e) contacting said extended first copies with said second set of primers to form more than one second bound entity;
f) extending said bound second set of primers by means of template sequences provided by said extended first copies to form more than one complex comprising extended first copies and extended second set of primers;
g) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;
h) hybridizing said nucleic acid copies formed in step g) to said array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of said hybridized copies obtained in step h).

468. The process of claim 467, wherein said nucleic acid array comprises members selected from the group consisting of DNA, RNA and analogs thereof.

469. The process of claim 468, wherein said analogs comprise PNA.

470. The process of claims 468 or 469, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

471. The process of claim 467, wherein said nucleic acid array is fixed or immobilized to a solid support.

472. The process of claim 471, wherein said solid support is porous or non-porous.

473. The process of claim 472, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

474. The process of claim 472, wherein said non-porous solid support comprises glass or plastic.

475. The process of claim 471, wherein said solid support is transparent, translucent, opaque or reflective.

476. The process of claim 471, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

477. The process of claim 471, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

478. The process of claim 467, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

479. The process of claim 467, wherein said library of nucleic acids analytes are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

480. The process of claim 467, wherein said first set of primers further comprise one or more sequences complementary to inherent universal detection targets (UDTs).

481. The process of claim 467, wherein said inherent UDT is selected from the group consisting of 3' poly A segments, consensus sequences, and a combination of both.

482. The process of claim 481, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

483. The process of claim 467, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

484. The process of claim 483, wherein said RNA promoters comprise phage promoters.

485. The process of claim 484, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

486. The process of claim 467, wherein said extending step d), the four or more non-inherent homopolymeric nucleotides are added by terminal transferase.

487. The process of claim 467, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

488. The process of claim 487, wherein said signaling entities generate a signal directly or indirectly.

489. The process of claim 488, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

490. The process of claim 489, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

491. The process of claim 490, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

492. The process of claim 467, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

493. The process of claim 467, wherein said means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

494. The process of claim 467, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

495. The process of claim 467, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

496. The process of claim 467, wherein step g) is carried out repeatedly.

497. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers and a second set of primers, wherein said first set comprises at least one production center;
(iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of said second set of primers;
and (v) means for ligating said set of oligonucleotides or polynucleotides (iv);
b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity;
c) extending said bound first set of primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;

d) ligating said set of oligonucleotides or polynucleotides a) (iv) to the 3' end of said first copies formed in step c) to form more than one ligated product;
e) contacting said ligated product with said second set of primers to form more than one second bound entity;
f) extending said bound second set of primers by means of template sequences provided by said ligated products formed in step d) to form more than one complex comprising said ligated products and said extended second set of primers;
g) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;
h) hybridizing said nucleic acid copies formed in step g) to said array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of said hybridized copies obtained in step h).

498. The process of claim 497, wherein said nucleic acid array comprises members selected from the group consisting of DNA, RNA and analogs thereof.

499. The process of claim 498, wherein said analogs comprise PNA.

500. The process of claims 498 or 499, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

501. The process of claim 497, wherein said nucleic acid array is fixed or immobilized to a solid support.

502. The process of claim 501, wherein said solid support is porous or non-porous.

503. The process of claim 502, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

504. The process of claim 502, wherein said non-porous solid support comprises glass or plastic.

505. The process of claim 501, wherein said solid support is transparent, translucent, opaque or reflective.

506. The process of claim 501, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

507. The process of claim 506, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

508. The process of claim 497, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

509. The process of claim 497, wherein said library of nucleic acids analytes are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

510. The process of claim 497, wherein said first set of primers are complementary to inherent universal detection targets (UDTs).

511. The process of claim 497, wherein said inherent UDTs are selected from the group consisting of 3' poly A segments, consensus sequences, and a combination of both.

512. The process of claim 511, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

513. The process of claim 497, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

514. The process of claim 513, wherein said RNA promoters comprise phage promoters.

515. The process of claim 514, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

516. The process of claim 497, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporate thereto.

517. The process of claim 516, wherein said signaling entities generate a signal directly or indirectly.

518. The process of claim 517, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

519. The process of claim 517, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

520. The process of claim 519, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

521. The process of claim 497, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

522. The process of claim 497, wherein said ligating means comprise T4 DNA
ligase.

523. The process of claim 497, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

524. The process of claim 497, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

525. The process of claim 497, wherein step g) is. carried out repeatedly.

526. The process of claim 497, wherein said means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

527. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers and a second set of primers, wherein said second set comprises at least one production center;
(iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of said second set of primers;
and (v) means for ligating said set of oligonucleotides or polynucleotides (iv);
b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity;
c) extending said bound first set of primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;
d) ligating said set of oligonucleotides or polynucleotides a) (iv) to the 3' end of said first copies formed in step c) to form more than one ligated product;
e) contacting said ligated product with said second set of primers to form more than one second bound entity;
f) extending said bound second set of primers by means of template sequences provided by said ligated products formed in step d) to form more than one complex comprising said ligated products and said extended second set of primers;
g) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;
h) hybridizing said nucleic acid copies formed in step g) to said array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of said hybridized copies obtained in step h).

528. The process of claim 527, wherein said nucleic acid array comprises members selected from the group consisting of DNA, RNA and analogs thereof.

529. The process of claim 528, wherein said analogs comprise PNA.

530. The process of claims 528 or 529, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

531. The process of claim 527, wherein said nucleic acid array is fixed or immobilized to a solid support.

532. The process of claim 531, wherein said solid support is porous or non-porous.

533. The process of claim 532, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

534. The process of claim 532, wherein said non-porous solid support comprises glass or plastic.

535. The process of claim 531, wherein said solid support is transparent, translucent, opaque or reflective.

536. The process of claim 531, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

537. The process of claim 536, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

538. The process of claim 527, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

539. The process of claim 527, wherein said library of nucleic acids analytes are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

540. The process of claim 527, wherein said first set of primers comprise one or more sequences which are complementary to inherent universal detection targets (UDTs).

541. The process of claim 527, wherein said inherent UDTs are selected from the group consisting of 3' poly A segments, consensus sequences, and a combination of both.

542. The process of claim 541, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

543. The process of claim 527, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

544. The process of claim 543, wherein said RNA promoters comprise phage promoters.

545. The process of claim 544, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

546. The process of claim 527, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

547. The process of claim 546, wherein said signaling entities generate a signal directly or indirectly.

548. The process of claim 547, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

549. The process of claim 547, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

550. The process of claim 549, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

551. The process of claim 527, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HlV-1 reverse transcriptase, HlV-2 reverse transcriptase, Sensiscript and Omniscript.

552. The process of claim 527, wherein said ligating means comprise T4 DNA
ligase.

553. The process of claim 527, wherein said means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

554. The process of claim 527, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

555. The process of claim 527, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

556. The process of claim 527, wherein step g) is carried out repeatedly.

557. The process of claim 527, wherein said means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

558. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers, a second set of primers and a third set of primers wherein said third set comprises at least one production center; and b) contacting said library of nucleic acid analytes with said first set of primers to form a first set of bound primers;
c) extending said first set of bound primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;
d) contacting said extended first copies with said second set of primers to form a second set of bound primers;
e) extending said second set of bound primers by means of template sequences provided by said extended first copies to form second copies of said nucleic acid analytes;
f) contacting said second copies with said third set of primers to form more than one third bound entity to form a third set of bound primers;
g) extending said third set of bound primers by means of template sequences provided by said extended second set of primers to form a hybrid comprising a second copy, a third copy and at least one production center;

h) synthesizing from said production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;
i) hybridizing said nucleic acid copies formed in step i) to said array of nucleic acids provided in step a) (i); and j) detecting or quantifying any of said hybridized copies obtained in step i).

559. The process of claim 558, wherein said nucleic acid array comprises members selected from the group consisting of DNA, RNA and analogs thereof.

560. The process of claim 559, wherein said analogs comprise PNA.

561. The process of claims 559 or 560, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

562. The process of claim 558, wherein said nucleic acid array is fixed or immobilized to a solid support.

563. The process of claim 562, wherein said solid support is porous or non-porous.

564. The process of claim 563, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

565. The process of claim 563, wherein said non-porous solid support comprises glass or plastic.

566. The process of claim 562, wherein said solid support is transparent, translucent, opaque or reflective.

567. The process of claim 562, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

568. The process of claim 562, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

569. The process of claim 558, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

570. The process of claim 558, wherein said library of nucleic acids analytes are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

571. The process of claim 558, wherein said first set of primers comprise one or more sequences which are complementary to inherent universal detection targets (UDTs).

572. The process of claim 558, wherein said inherent UDTs are selected from the group consisting of 3' poly A segments, consensus sequences, and a combination of both.

573. The process of claim 572, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

574. The process of claim 558, wherein said second set of primers are random primers.

575. The process of claim 558, further comprising the step c') of adding a primer binding site after step c).

576. The process of claim 575, wherein said second set of primers are complementary to said primer binding site.

577. The process of claim 575, wherein said primer binding site is added by means of T4 DNA ligase or terminal transferase.

578. The process of claim 558, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

579. The process of claim 578, wherein said RNA promoters comprise phage promoters.

580. The process of claim 579, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

581. The process of claim 558, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

582. The process of claim 581, wherein said signaling entities generate a signal directly or indirectly.

583. The process of claim 582, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

584. The process of claim 582, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

585. The process of claim 584, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

586. The process of claim 558, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

587. The process of claim 558, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

588. The process of claim 558, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

589. The process of claim 558, wherein step g) is carried out repeatedly.

590. The process of claim 558, further comprising the step f') of separating said extended second set of primers obtained in step e).

591. The process of claim 558, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

592. The process of claim 558, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

593. The process of claim 558, wherein step g) is carried out repeatedly.

594. The process of claim 558, wherein said means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

595. The process of claim 594, wherein said second set of primers comprise at least one production center which differs in nucleotide sequence from said production center in the third set of primers.

596. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers and a second set of primers, wherein said first set of primers are fixed or immobilized to a solid support, and wherein said second set of primers comprises at least two segments, the first segment at the 3' end comprising random sequences, and the second segment comprising at least one production center;
(iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions;
b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity;
c) extending said bound first set of primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;
d) contacting said extended first copies with said second set of primers to form more than one second bound entity;
e) extending said bound second set of primers by means of template sequences provided by said extended first copies to form more than one complex comprising extended first copies and extended second set of primers;

f) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;

g) hybridizing said nucleic acid copies formed in step f) to said array of nucleic acids provided in step a) (i); and h) detecting or quantifying any of said hybridized copies obtained in step g).

597. The process of claim 596, wherein said solid support comprises beads.

598. The process of claim 597, wherein said beads are magnetic.

599. The process of claim 596, wherein said nucleic acid array comprises members selected from the group consisting of DNA, RNA and analogs thereof.

600. The process of claim 599, wherein said analogs comprise PNA.

601. The process of claims 599 or 600, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

602. The process of claim 596, wherein said nucleic acid array is fixed or immobilized to a solid support.

603. The process of claim 602, wherein said solid support is porous or non-porous.

604. The process of claim 603, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

605. The process of claim 603, wherein said non-porous solid support comprises glass or plastic.

606. The process of claim 602, wherein said solid support is transparent, translucent, opaque or reflective.

607. The process of claim 602, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

608. The process of claim 607, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

609. The process of claim 596, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

610. The process of claim 596, wherein said library of nucleic acids analytes are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

611. The process of claim 596, wherein said first set of primers comprise one or more sequences which are complementary to inherent universal detection targets (UDTs).

612. The process of claim 596, wherein said inherent UDTs are selected from the group consisting of 3' poly A segments, consensus sequences, and a combination of both.

613. The process of claim 612, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

614. The process of claim 596, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

615. The process of claim 614, wherein said RNA promoters comprise phage promoters.

616. The process of claim 615, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

617. The process of claim 596, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

618. The process of claim 617, wherein said signaling entities generate a signal directly or indirectly.

619. The process of claim 618, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

620. The process of claim 618, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

621. The process of claim 620, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

622. The process of claim 596, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol l, Klenow fragment of E. coli DNA
Pol l, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HlV-1 reverse transcriptase, HlV-2 reverse transcriptase, Sensiscript and Omniscript.

623. The process of claim 596, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

624. The process of claim 596, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

625. The process of claim 596, wherein step g) is carried out repeatedly.

626. The process of claim 596, wherein said means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

627. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers and a second set of primers, wherein said first set of primers are fixed or immobilized to a solid support, and wherein said first set of primers comprise at least one production center; and (iv) means for synthesizing nucleic acid copies under isothermal or isostatic conditions;
b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity;
c) extending said bound first set of primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;
d) extending said first copies by means of at least four (4) or more non-inherent homopolymeric nucleotides;
e) contacting said extended first copies with said second set of primers to form more than one second bound entity;
f) extending said bound second set of primers by means of template sequences provided by said extended first copies to form more than one complex comprising extended first copies and extended second set of primers;
g) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;
h) hybridizing said nucleic acid copies formed in step g) to said array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of said hybridized copies obtained in step h).

628. The process of claim 627, wherein said solid support comprises beads.

629. The process of claim 628, wherein said beads are magnetic.

630. The process of claim 627, wherein said nucleic acid array comprises members selected from the group consisting of DNA, RNA and analogs thereof.

631. The process of claim 630, wherein said analogs comprise PNA.

632. The process of claims 630 or 631, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

633. The process of claim 627, wherein said nucleic acid array is fixed or immobilized to a solid support.

634. The process of claim 633, wherein said solid support is porous or non-porous.

635. The process of claim 634, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

636. The process of claim 634, wherein said non-porous solid support comprises glass or plastic.

637. The process of claim 633, wherein said solid support is transparent, translucent, opaque or reflective.

638. The process of claim 633, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

639. The process of claim 638, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

640. The process of claim 627, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

641. The process of claim 627, wherein said library of nucleic acids analytes are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

642. The process of claim 627, wherein said first set of primers further comprise one or more sequences complementary to inherent universal detection targets (UDTs).

643. The process of claim 627, wherein said inherent UDT is selected from the group consisting of 3' poly A segments, consensus sequences, and a combination of both.

644. The process of claim 643, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

645. The process of claim 627, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

646. The process of claim 645, wherein said RNA promoters comprise phage promoters.

647. The process of claim 646, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

648. The process of claim 627, wherein said extending step d), the four or more non-inherent homopolymeric nucleotides are added by terminal transferase.

649. The process of claim 627, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

650. The process of claim 649, wherein said signaling entities generate a signal directly or indirectly.

651. The process of claim 650, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

652. The process of claim 650, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

653. The process of claim 652, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

654. The process of claim 627, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

655. The process of claim 627, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

656. The process of claim 627, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

657. The process of claim 627, wherein step g) is carried out repeatedly.

658. The process of claim 627, wherein said means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

659. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers and a second set of primers, wherein said first set of primers are fixed or immobilized to a solid support, and wherein said first set comprises at least one production center;
(iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of said second set of primers;
and (v) means for ligating said set of oligonucleotides or polynucleotides (iv);
b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity;
c) extending said bound first set of primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;
d) ligating said set of oligonucleotides or polynucleotides a) (iv) to the 3' end of said first copies formed in step c) to form more than one ligated product;
e) contacting said ligated product with said second set of primers to form more than one second bound entity;
f) extending said bound second set of primers by means of template sequences provided by said ligated products formed in step d) to form more than one complex comprising said ligated products and said extended second set of primers;
g) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;
h) hybridizing said nucleic acid copies formed in step g) to said array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of said hybridized copies obtained in step h).

660. The process of claim 659, wherein said solid support comprises beads.

661. The process of claim 660, wherein said beads are magnetic.

662. The process of claim 659, wherein said nucleic acid array comprises members selected from the group consisting of DNA, RNA and analogs thereof.

663. The process of claim 662, wherein said analogs comprise PNA.

664. The process of claims 662 or 663, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

665. The process of claim 659, wherein said nucleic acid array is fixed or immobilized to a solid support.

666. The process of claim 665, wherein said solid support is porous or non-porous.

667. The process of claim 666, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

668. The process of claim 666, wherein said non-porous solid support comprises glass or plastic.

669. The process of claim 665, wherein said solid support is transparent, translucent, opaque or reflective.

670. The process of claim 665, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

671. The process of claim 665, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

672. The process of claim 659, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

673. The process of claim 659, wherein said library of nucleic acids analytes are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

674. The process of claim 659, wherein said first set of primers comprise one or more sequences which are complementary to inherent universal detection targets (UDTs).

675. The process of claim 659, wherein said inherent UDTs are selected from the group consisting of 3' poly A segments, consensus sequences, and a combination of both.

676. The process of claim 675, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

677. The process of claim 659, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

678. The process of claim 677, wherein said RNA promoters comprise phage promoters.

679. The process of claim 678, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

680. The process of claim 659, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

681. The process of claim 680, wherein said signaling entities generate a signal directly or indirectly.

682. The process of claim 681, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

683. The process of claim 682, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

684. The process of claim 683, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

685. The process of claim 659, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

686. The process of claim 659, wherein said ligating means comprise T4 DNA
ligase.

687. The process of claim 659, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

688. The process of claim 659, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

689. The process of claim 659, wherein step g) is carried out repeatedly.

690. The process of claim 659, wherein said means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

691. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers and a second set of primers, wherein said first set of primers are fixed or immobilized to a solid support, and wherein said second set comprises at least one production center;

(iv) a set of oligonucleotides or polynucleotides complementary to at least one segment or sequence of said second set of primers;

and (v) means for ligating said set of oligonucleotides or polynucleotides (iv);

b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity;

c) extending said bound first set of primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;

d) ligating said set of oligonucleotides or polynucleotides a) (iv) to the 3' end of said first copies formed in step c) to form more than one ligated product;

e) contacting said ligated product with said second set of primers to form more than one second bound entity;

f) extending said bound second set of primers by means of template sequences provided by said ligated products formed in step d) to form more than one complex comprising said ligated products and said extended second set of primers;

g) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;

h) hybridizing said nucleic acid copies formed in step g) to said array of nucleic acids provided in step a) (i); and i) detecting or quantifying any of said hybridized copies obtained in step h).

692. The process of claim 691, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

693. The process of claim 691, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

694. The process of claim 691, wherein step g) is carried out repeatedly.

695. The process of claim 691, wherein said means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

696. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of fixed or immobilized nucleic acids identical or complementary in part or whole to sequences of said nucleic acids of interest;

(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes, said polymerizing means comprising a first set of primers, a second set of primers and a third set of primers, wherein said first set of primers are fixed or immobilized to a solid support, and wherein said third set comprises at least one production center; and b) contacting said library of nucleic acid analytes with said first set of primers to form more than one first bound entity;

c) extending said bound first set of primers by means of template sequences provided by said nucleic acid analytes to form first copies of said analytes;
d) contacting said extended first copies with said second set of primers to form more than one second bound entity;

e) extending said bound second set of primers by means of template sequences provided by said extended first copies to form an extended second set of primers;

f) separating said extended second set of primers obtained in step e) g) contacting said extended second set of primers with said third set of primers to form more than one third bound entity;

h) extending said third bound entity by means of template sequences provided by said extended second set of primers to form more than one complex comprising said extended third bound entity and said extended set of primers;

i) synthesizing from a production center in said second set of primers in said complexes one or more nucleic acid copies under isothermal or isostatic conditions;

j) hybridizing said nucleic acid copies formed in step i) to said array of nucleic acids provided in step a) (i); and k) detecting or quantifying any of said hybridized copies obtained in step j).

697. The process of claim 696, wherein said solid support comprises beads.

698. The process of claim 697, wherein said beads are magnetic.

699. The process of claim 696, wherein said nucleic acid array comprises members selected from the group consisting of DNA, RNA and analogs thereof.

700. The process of claim 699, wherein said analogs comprise PNA.

701. The process of claims 699 or 700, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

702. The process of claim 696, wherein said nucleic acid array is fixed or immobilized to a solid support.

703. The process of claim 702, wherein said solid support is porous or non-porous.

704. The process of claim 703, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

705. The process of claim 703, wherein said non-porous solid support comprises glass or plastic.

706. The process of claim 703, wherein said solid support is transparent, translucent, opaque or reflective.

707. The process of claim 703, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

708. The process of claim 707, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

709. The process of claim 696, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

710. The process of claim 696, wherein said library of nucleic acids analytes are derived from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

711. The process of claim 696, wherein said first set of primers comprise one or more sequences which are complementary to inherent universal detection targets (UDTs).

712. The process of claim 696, wherein said inherent UDTs are selected from the group consisting of 3' poly A segments, consensus sequences, and a combination of both.

713. The process of claim 712, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

714. The process of claim 696, wherein said second set of primers are random primers.

715. The process of claim 696, further comprising the step c') of adding a primer binding site after step c).

716. The process of claim 715, wherein said second set of primers are complementary to said primer binding site.

717. The process of claim 715, wherein said primer binding site is added by means of T4 DNA ligase or terminal transferase.

718. The process of claim 696, wherein said production center is selected from the group consisting of primer binding sites, RNA promoters, or a combination of both.

719. The process of claim 718, wherein said RNA promoters comprise phage promoters.

720. The process of claim 719, wherein said phage promoters are selected from the group consisting of T3, T7 and SP6.

721. The process of claim 696, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

722. The process of claim 721, wherein said signaling entities generate a signal directly or indirectly.

723. The process of claim 722, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

724. The process of claim 722, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

725. The process of claim 724, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

726. The process of claim 696, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol l, Klenow fragment of E. coli DNA
Pol l, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerise, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

727. The process of claim 696, further comprising the step of separating the first copies obtained from step c) from their templates and repeating step b).

728. The process of claim 696, further comprising the step of separating the extended second set of primers obtained from step f) from their templates and repeating step e).

729. The process of claim 696, wherein step g) is carried out repeatedly.

730. The process of claim 696, wherein said means for synthesizing nucleic acid copies under isothermal or isostatic conditions is carried out by one or more members selected from the group consisting of RNA transcription, strand displacement amplification and secondary structure amplification.

731. The process of claim 696, wherein said second set of primers comprise at least one production center which differs in nucleotide sequence from said production center in the third set of primers.

732. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified; and (iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes said polymerizing means comprising a first set of primers;
b) contacting said nucleic acid analytes with said first set of primers to form a first bound entity;
c) extending said bound set of first set of primers by means of template sequences provided by said nucleic acid analytes to form first nucleic acid copies of said analytes;
d) separating said first nucleic acid copies from the said analytes;
e) repeating steps b), c) and d) until a desirable amount of first nucleic acid copies have been synthesized;
f) hybridizing said nucleic nucleic acid copies formed in step e) to said array of nucleic acids provided in step (i); and g) detecting or quantifying any of said hybridized first nucleic acid copies obtained in step f).

733. The process of claim 732, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

734. The process of claim 4, wherein said analogs comprise PNA.

735. The process of claims 733 or 734, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

736. The process of claim 732, wherein said array of nucleic acids are fixed or immobilized to a solid support.

737. The process of claim 736, wherein said solid support is porous or non-porous.

738. The process of claim 737, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

739. The process of claim 736, wherein said non-porous solid support comprises glass or plastic.

740. The process of claim 736, wherein said solid support is transparent, translucent, opaque or reflective.

741. The process of claim 736, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

742. The process of claim 741, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

743. The process of claim 732, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

744. The process of claim 732, wherein said nucleic acid analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

745. The process of claim 732, wherein said nucleic acid analytes comprise an inherent UDT selected from the group consisting of poly T segments, secondary structures, consensus sequences, and a combination of any of the foregoing.

746. The process of claim 745, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

747. The process of claim 732, further comprising the step of adding one or more non-inhererent UDTs to said nucleic acid analytes or said first copies by an enzymatic means selected from the group consisting of poly A polymerase, terminal transferase, T4 DNA ligase, T4 RNA ligase and a combination of any of the foregoing.

748. The process of claim 732, wherein said providing or contacting steps, the first set of primers comprise one or more UDTs.

749. The process of claim 732, wherein said polymerizing means comprises an enzyme selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA
polymerase, Tth DNA Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

750. The process of claim 749, wherein an additional amount of enzyme is added after step d) or after repeating step d).

751. The process of claim 732, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

752. The process of claim 748, wherein said UDE generates a signal directly or indirectly.

753. The process of claim 752, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound; an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

754. The process of claim 752, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

755. The process of claim 754, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

756. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing (i) an array of fixed or immobilized nucleic acids identical in part or whole to sequences of said nucleic acids of interest;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest sought to be detected or quantified;
(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acid analytes said polymerizing means comprising a first set of primers and a second set of primers;
(iv) means for addition of sequences to the 3' end of nucleic acids;
b) contacting said nucleic acid analytes with said first set of primer to form a first bound entity;
c) extending said bound set of first set of primers by means of template sequences provided by said nucleic acid analytes to form first nucleic acid copies of-said analytes;
d) extending said first nucleic copies by the addition of non-template derived sequences to the 3' end of said first nucleic acid copies e) contacting said extended first nucleic acid copies with said second set of primers to form a second bound entity;
f) extending said bound set of second set of primers by means of template sequences provided by said extended first nucleic acid copies to form second nucleic acid copies;
g) separating said second nucleic acid copies from the extended first nucleic acid copies;
h) repeating steps e), f) and g) until a desirable amount of second nucleic acid copies have been synthesized;
i) hybridizing said second nucleic acid copies formed in step h) to said array of nucleic acids provided in step (i); and j) detecting or quantifying any of said hybridized second nucleic acid copies obtained in step i).

757. The process of claim 756, wherein said nucleic acid array is selected from the group consisting of DNA, RNA and analogs thereof.

758. The process of claim 757, wherein said analogs comprise PNA.

759. The process of claims 757 or 758, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

760. The process of claim 756, wherein said solid support is porous or non-porous.

761. The process of claim 760, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

762. The process of claim 760, wherein said non-porous solid support comprises glass or plastic.

763. The process of claim 758, wherein said solid support is transparent, translucent, opaque or reflective.

764. The process of claim 756, wherein said nucleic acids are directly or indirectly fixed or immobilized to said solid support.

765. The process of claim 764, wherein said nucleic acids are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

766. The process of claim 758, wherein said library of nucleic acid analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

767. The process of claim 758, wherein said nucleic acid analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA and a combination of any of the foregoing.

768. The process of claim 758, wherein said nucleic acid analytes comprise an inherent UDT selected from the group consisting of poly T segments, secondary structures, consensus sequences, and a combination of any of the foregoing.

769. The process of claim 768, wherein said consensus sequences is selected from the group consisting of signal sequences for poly A addition, splicing elements, multicopy repeats, and a combination of any of the foregoing.

770. The process of claim 758, further comprising the step of adding one or more non-inhererent UDTs to said nucleic acid analyzes, said first copies or said second copies by an enzymatic means selected from the group consisting of poly A
polymerase, terminal transferase, T4 DNA ligase, T4 RNA ligase and a combination of any of the foregoing.

771. The process of claim 758, wherein said providing or contacting steps, the first set of primers or the second set of primers or both comprise one or more UDTs.

772. The process of claim 758, wherein said extending step d) is carried out by an enzymatic means selected from the group consisting of terminal transferase, DNA ligase, T4 RNA ligase, and a combination of any of the foregoing.

773. The process of claim 758, wherein said polymerizing means comprises an enzyme selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA
polymerase, Tth DNA Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

774. The process of claim 773, wherein following one or more separation steps an additional amount of enzyme is added.

775. The process of claim 758, wherein said hybridized nucleic acid copies further comprise one or more signaling entities attached or incorporated thereto.

776. The process of claim 775, wherein said signaling entities generate a signal directly or indirectly.

777. The process of claim 776, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

778. The process of claim 776, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

779. The process of claim 778, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

780. The process of claim 756, which comprises the additional steps of k) separating the first nucleic copies produced in step c) of claim 1750 from said analytes ~) repeating steps b) c) and k) until a desirable amount of first nucleic acid copies have been synthesized

781. The process of claim 756, which comprises the additional steps of l) separating the extended first nucleic copies produced in step d) of claim 1750 from said analytes and m) repeating steps b), c), d) and l) until a desirable amount of extended first nucleic acid copies have been synthesized.

782. The process of claim 756, wherein said first set of primers are attached to a solid support.

783. The process of claim 782, wherein said solid support comprises beads.

784. The process of claim 783, wherein said beads are magnetic.

785. A composition of matter that comprises an array of solid surfaces comprising discrete areas;
wherein at least two of said discrete areas each comprises:
a first set of nucleic acid primers; and a second set of nucleic acid primers;
wherein the nucleotide sequences in said first set of nucleic acid primers are different from the nucleotide sequences in said second set o,f nucleic acid primers;

wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical.

786. The composition of claim 785, wherein said array of solid surfaces has been designed/synthesized such that D1 is less than D2, said D1 being the physical distance on said array between a nucleic acid primer that is part of a first set of an area and the nucleic acid primer is part of a second set of the same area, and being the physical distance in a nucleic acid in a sample between the sequence of a primer binding site in said nucleic acid in a sample for the nucleic acid primer of the first set and the complement of the primer binding site in the said nucleic acid in the sample for the nucleic acid primer in the second set.

787. The composition of claim 785, wherein said nucleic acid primers are selected from the group consisting of DNA, RNA and analogs thereof.

788. The composition of claim 787, wherein said analogs comprise PNA.

789. The composition of claims 787 or 788, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

790. The composition of claim 785, wherein said solid surfaces are porous or non-porous.

791. The composition of claim 789, wherein said porous solid surfaces are selected from the group consisting of polyacrylamide and agarose.

792. The composition of claim 790, wherein said non-porous solid surfaces comprise glass or plastic.

793. The composition of claim 785, wherein said solid surfaces are transparent, translucent, opaque or reflective.

794. The composition of claim 785, wherein nucleic acid primers are directly or indirectly fixed or immobilized to said solid surfaces.

795. The composition of claim 794, wherein said nucleic acid primers are indirectly fixed or immobilized to said solid surfaces by means of a chemical linker or linkage arm.

796. A composition of matter that comprises an array of solid surfaces comprising a plurality of discrete areas;
wherein at least two of said discrete areas each comprises:
a first set of nucleic acid primers; and a second set of nucleic acid primers;
wherein the nucleotide sequences in said first set of nucleic acid primers are different from the nucleotide sequences in said second set of nucleic acid primers;
wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ substantially from each other; and wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical.

797. The composition of claim 795, wherein said array of solid surfaces has been designed/synthesized such that D1 is less than D2, said D1 being the physical distance on said array between a nucleic acid primer that is part of a first set of an area and the nucleic acid primer is part of a second set of the same area, and being the physical distance in a nucleic acid in a sample between the sequence of a primer binding site in said nucleic acid in a sample for the nucleic acid primer of the first set and the complement of the primer binding site in the said nucleic acid in the sample for the nucleic acid primer in the second set.

798. The composition of claim 796, wherein said nucleic acid primers are selected from the group consisting of DNA, RNA and analogs thereof.

799. The composition of claim 798, wherein said analogs comprise PNA.

800. The composition of claims 798 or 799, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

801. The composition of claim 796, wherein said solid surfaces are porous or non-porous.

802. The composition of claim 801, wherein said porous solid surfaces are selected from the group consisting of polyacrylamide and agarose.

803. The composition of claim 796, wherein said non-porous solid surfaces comprise glass or plastic.

804. The composition of claim 796, wherein said solid surfaces are transparent, translucent, opaque or reflective.

805. The composition of claim 796, wherein nucleic acid primers are directly or indirectly fixed or immobilized to said solid surfaces.

806. The composition of claim 805, wherein said nucleic acid primers are indirectly fixed or immobilized to said solid surfacees by means of a chemical linker or linkage arm.

807. A process for producing two or more copies of nucleic acids of interest in a library comprising the steps of:
a) providing:
(i) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of said discrete areas each comprises:
(1) a first set of nucleic acid primers; and (2) a second set of nucleic acid primers;
wherein the nucleotide sequences in said first set of nucleic acid primers are different from the nucleotide sequences in said second set of nucleic acid primers;
wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical;
(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest;

(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acids of interest;
b) contacting a primer of said first set with a complementary sequence in said nucleic acid of interest;
c) extending said primer in the first set using said nucleic acid of interest as a template to generate an extended first primer;
d) contacting a, primer in said second set with a complementary sequence in said extended first primer;
e) extending said primer in the second set using said extended first primer as a template to generate an extended second primer;
f) contacting a primer in the first set with a complementary sequence in said extended second primer;
g) extending said primer in the first set using said extended second primer as a template to generate an extended first primer; and h) repeating steps d) through g) above one or more times.

808. The process of claim 807, wherein said nucleic acid primers are selected from the group consisting of DNA, RNA and analogs thereof.

809. The process of claim 808, wherein said analogs comprise PNA.

810. The process of claims 808 or 809, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

811. The process of claim 807, wherein said solid support is porous or non-porous.

812. The process of claim 811, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

813. The process of claim 811, wherein said non-porous solid support comprises glass or plastic.

814. The process of claim 807, wherein said solid support is transparent, translucent, opaque or reflective.

815. The process of claim 807, wherein nucleic acid primers are directly or indirectly fixed or immobilized to said solid support.

816. The process of claim 815, wherein said nucleic acid primers are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

817. The process of claim 807, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

818. The process of claim 807, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

819. The process of claim 600, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

820. A process for detecting or quantifying more than one nucleic acid of interest in a library comprising the steps of:
a) providing:
(i) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of said discrete areas each comprises:
(1) a first set of nucleic acid primers; and (2) a second set of nucleic acid primers;
wherein the nucleotide sequences in said first set of nucleic acid primers are different from the nucleotide sequences in said second set of nucleic acid primers;
wherein the nucleotide sequences of a first set of nucleic acid primers of a first discrete area and the nucleotide sequences of a first set of nucleic acid primers of a second discrete area differ from each other by at least one base; and wherein the nucleotide sequences of the second set of nucleic acid primers of a first discrete area and the nucleotide sequences of the second set of nucleic acid primers of a second discrete area are substantially the same or identical;

(ii) a library of nucleic acid analytes which may contain the nucleic acids of interest;
(iii) polymerizing means for synthesizing nucleic acid copies of said nucleic acids of interest; and (iv) non-radioactive signal generating means capable of being attached to or incorporated into nucleic acids;
b) contacting a primer of said first set with a complementary sequence in said nucleic acid of interest;
c) extending said primer in the first set using said nucleic acid of interest as a template to generate an extended first primer;
d) contacting a primer in said second set with a complementary sequence in said extended first primer;
e) extending said primer in the second set using said extended first primer as a template to generate an extended second primer;
f) contacting a primer in the first set with a complementary sequence in said extended second primer;
g) extending said primer in the first set using said extended second primer as a template to generate an extended first primer;
h) repeating steps d) through g) above one or more times; and i) detecting or quantifying by means of said non-radioactive signal generating means attached to or incorporated into any of said extended primers in steps c), e), g), and h).

821. The process of claim 820, wherein said nucleic acid primers are selected from the group consisting of DNA, RNA and analogs thereof.

822. The process of claim 821, wherein said analogs comprise PNA.

823. The process of claims 821 or 822, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

824. The process of claim 820, wherein said solid support is porous or non-porous.

825. The process of claim 824, wherein said porous solid support is selected from the group consisting of polyacrylamide and agarose.

826. The process of claim 824, wherein said non-porous solid support comprises glass or plastic.

827. The process of claim 820, wherein said solid support is transparent, translucent, opaque or reflective.

828. The process of claim 820, wherein nucleic acid primers are directly or indirectly fixed or immobilized to said solid support.

829. The process of claim 828, wherein said nucleic acid primers are indirectly fixed or immobilized to said solid support by means of a chemical linker or linkage arm.

830. The process of claim 820, wherein said library of analytes is derived from a biological source selected from the group consisting of organs, tissues and cells.

831. The process of claim 820, wherein said analytes are selected from the group consisting of genomic DNA, episomal DNA, unspliced RNA, mRNA, rRNA, snRNA
and a combination of any of the foregoing.

832. The process of claim 820, wherein said polymerizing means are selected from the group consisting of E. coli DNA Pol I, Klenow fragment of E. coli DNA
Pol I, Bst DNA polymerase, Bca DNA polymerase, Taq DNA polymerase, Tth DNA
Polymerase, T4 DNA polymerase, ALV reverse transcriptase, MuLV reverse transcriptase, RSV reverse transcriptase, HIV-1 reverse transcriptase, HIV-2 reverse transcriptase, Sensiscript and Omniscript.

833. The process of claim 820, wherein said non-radioactive signal generating means are selected from the group consisting of labeled nucleotides, intercalating dyes, universal detection elements and a combination of any of the foregoing.

834. The process of claim 820, wherein said extended primers further comprise one or more signaling entities attached or incorporated thereto.

835. The process of claim 834, wherein said signaling entities generate a signal directly or indirectly.

836. The process of claim 835, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

837. The process of claim 835, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

838. The process of claim 837, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

839. A composition of matter that comprises an array of solid surfaces comprising a plurality of discrete areas;
wherein at least two of said discrete areas comprise:
a chimeric composition comprising:
a nucleic acid portion; and a non-nucleic acid portion;
wherein said nucleic acid portion of a first discrete area has the same sequence as the nucleic acid portion of a second discrete area; and wherein said non-nucleic acid portion has a binding affinity for analytes of interest.

840. The composition of claim 839, wherein said nucleic acid portion is selected from the group consisting of DNA, RNA and analogs thereof.

841. The composition of claim 840, wherein said analogs comprise PNA.

842. The composition of claims 840 or 841, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

843. The composition of claim 839, wherein said solid surfaces are porous or non-porous.

844. The composition of claim 843, wherein said porous solid surfaces are selected from the group consisting of polyacrylamide and agarose.

845. The composition of claim 843, wherein said non-porous solid surfaces comprise glass or plastic.

846. The composition of claim 839, wherein said solid surfaces are transparent, translucent, opaque or reflective.

847. The composition of claim 839, wherein said nucleic acid portions are directly or indirectly fixed or immobilized to said solid surfaces.

848. The composition of claim 839, wherein said non-nucleic acid portions are selected from the group consisting of peptides, proteins, ligands, enzyme substrates, hormones, receptors, drugs and a combination of any of the foregoing.

849. A composition of matter that comprises an array of solid surfaces comprising a plurality of discrete areas;
wherein at least two of said discrete areas comprise:
a chimeric composition hybridized to complementary sequences of nucleic acids fixed or immobilized to said discrete areas, wherein said chimeric composition comprises:
a nucleic acid portion; and a non-nucleic acid portion;
said nucleic acid portion comprising at least one sequence, wherein said non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when said non-nucleic acid portion is a peptide or protein, said nucleic acid portion does not comprises sequences which are either identical or complementary to sequences that code for said peptide or protein.

850. The composition of claim 849, wherein said solid surfaces are porous or non-porous.

851. The composition of claim 850, wherein said porous solid surfaces are selected from the group consisting of polyacrylamide and agarose.

852. The composition of claim 850, wherein said non-porous solid surfaces comprises glass or plastic.

853. The composition of claim 849, wherein said solid surfaces are transparent, translucent, opaque or reflective.

854. The composition of claim 849, wherein said fixed or immobilized nucleic acid is selected from the group consisting of DNA, RNA and analogs thereof.

855. The composition of claim 854, wherein said analogs comprise PNA.

856. The composition of claims 854 or 855, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

857. The composition of claim 849, wherein said nucleic acid portion is selected from the group consisting of DNA, RNA and analogs thereof.

858. The composition of claim 857, wherein said analogs comprise PNA.

859. The composition of claims 857 or 858, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

860. The composition of claim 849, wherein said non-nucleic acid portions are selected from the group consisting of peptides, proteins, ligands, enzyme substrates, hormones, receptors, drugs and a combination of any of the foregoing.

861. A process for detecting or quantifying analytes of interest, said process comprising the steps of:
1) providing.
a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of said discrete areas comprise a chimeric composition comprising a nucleic acid portion; and a non-nucleic acid portion; wherein said nucleic acid portion of a first discrete area has the same sequence as the nucleic acid portion of a second discrete area; and wherein said non-nucleic acid portion has a binding affinity for analytes of interest;
b) a sample containing or suspected of containing one or more of said analytes of interest; and c) signal generating means;
2) contacting said array a) with the sample b) under conditions permissive of binding said analytes to said non-nucleic acid portion;
3) contacting said bound analytes with said signal generating means; and 4) detecting or quantifying the presence of said analytes.

862. The process of claim 861, wherein said solid surfaces are porous or non-porous.

863. The process of claim 862, wherein said porous solid surfaces are selected from the group consisting of polyacrylamide and agarose.

864. The process of claim 862, wherein said non-porous solid surfaces comprise glass or plastic.

865. The process of claim 861, wherein said solid surfaces are transparent, translucent, opaque or reflective.

866. The process of claim 861, wherein said nucleic acid portion is selected from the group consisting of DNA, RNA and analogs thereof.

867. The process of claim 866, wherein said analogs comprise PNA.

868. The process of claims 866 or 867, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

869. The process of claim 861, wherein said nucleic acid portions are directly or indirectly fixed or immobilized to said solid surfaces.

870. The process of claim 861, wherein said non-nucleic acid portions are selected from the group consisting of peptides, proteins, ligands, enzyme substrates, hormones, receptors, drugs and a combination of any of the foregoing.

871. The process of claim 861, wherein said signal generating means comprise direct signal generating means and indirect signal generating means.

872. The process of claim 871, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

873. The process of claim 871, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

874. The process of claim 873, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

875. A process for detecting or quantifying analytes of interest, said process comprising the steps of:
1) providing:
a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of said discrete areas comprise a chimeric composition comprising a nucleic acid portion; and a non-nucleic acid portion; wherein said nucleic acid portion of a first discrete area has the same sequence as the nucleic acid portion of a second discrete area; and wherein said non-nucleic acid portion has a binding affinity for analytes of interest;

b) a sample containing or suspected of containing one or more of said analytes of interest; and c) signal generating means;

2) labeling said analytes of interest with said signal generating means;

3) contacting said array a) with said labeled analytes under conditions permissive of binding said labeled analytes to said non-nucleic acid portion;
and 4) detecting or quantifying the presence of said analytes.

876. The process of claim 875, wherein said solid surfaces are porous or non-porous.

877. The process of claim 876, wherein said porous solid surfaces are selected from the group consisting of polyacrylamide and agarose.

878. The process of claim 876, wherein said non-porous solid surfaces comprise glass or plastic.

879. The process of claim 876, wherein said solid surfaces are transparent, translucent, opaque or reflective.

880. The process of claim 875, wherein said nucleic acid portion is selected from the group consisting of DNA, RNA and analogs thereof.

881. The process of claim 880, wherein said analogs comprise PNA.

882. The process of claims 880 or 881, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

883. The process of claim 875, wherein said nucleic acid portions are directly or indirectly fixed or immobilized to said solid surfaces.

884. The process of claim 875, wherein said non-nucleic acid portions are selected from the group consisting of peptides, proteins, ligands, enzyme substrates, hormones, receptors, drugs and a combination of any of the foregoing.

885. The process of claim 875, wherein said signal generating means comprise direct signal generating means and indirect signal generating means.

886. The process of claim 885, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

887. The process of claim 885, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

888. The process of claim 887, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

889. A process for detecting or quantifying analytes of interest, said process comprising the steps of:

1) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of said discrete areas comprise nucleic acids fixed or immobilized to said discrete areas, b) chimeric compositions comprising:

i) a nucleic acid portion; and ii) a non-nucleic acid portion;

said nucleic acid portion comprising at least one sequence, wherein said non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when said non-nucleic acid portion is a peptide or protein, said nucleic acid portion does not comprise sequences which are either identical or complementary to sequences that code for said peptide or protein;

c) a sample containing or suspected of containing said analytes of interest; and d) signal generating means;

2) contacting said array with said chimeric compositions to hybridize the nucleic acid portions of said chimeric compositions to complementary nucleic acids fixed or immobilized to said array;

3) contacting said array a) with the sample b) under conditions permissive of binding said analytes to said non-nucleic acid portion;

4) contacting said bound analytes with said signal generating means; and 5) detecting or quantifying the presence of said analytes.

890. The process of claim 889, wherein said solid surfaces are porous or non-porous.

891. The process of claim 890, wherein said porous solid surfaces are selected from the group consisting of polyacrylamide and agarose.

892. The process of claim 890, wherein said non-porous solid surfaces comprises glass or plastic.

893. The process of claim 889, wherein said solid surfaces are transparent, translucent, opaque or reflective.

894. The process of claim 889, wherein said fixed or immobilized nucleic acid is selected from the group consisting of DNA, RNA and analogs thereof.

895. The process of claim 894, wherein said analogs comprise PNA.

896. The process of claims 894 or 895, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

897. The process of claim 889, wherein said nucleic acid portion is selected from the group consisting of DNA, RNA and analogs thereof.

898. The process of claim 897, wherein said analogs comprise PNA.

899. The process of claims 897 or 898, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

900. The process of claim 889, wherein said non-nucleic acid portions are selected from the group consisting of peptides, proteins, ligands, enzyme substrates, hormones, receptors, drugs and a combination of any of the foregoing.

901. The process of claim 889, wherein said signal generating means comprise direct signal generating means and indirect signal generating means.

902. The process of claim 901, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

903. The process of claim 901, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

904. The process of claim 903, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

905. A process for detecting or quantifying analytes of interest, said process comprising the steps of:

1) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of said discrete areas comprise nucleic acids fixed or immobilized to said discrete areas, b) chimeric compositions comprising:
i) a nucleic acid portion; and ii) a non-nucleic acid portion;

said nucleic acid portion comprising at least one sequence, wherein said non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when said non-nucleic acid portion is a peptide or protein, said nucleic acid portion does not comprise sequences which are either identical or complementary to sequences that code for said peptide or protein;
c) a sample containing or suspected of containing said analytes of interest; and d) signal generating means;

2) contacting said chimeric compositions with the sample b) under conditions permissive of binding said analytes to said non-nucleic acid portion;

3) contacting said array with said chimeric compositions to hybridize the nucleic acid portions of said chimeric compositions to complementary nucleic acids fixed or immobilized to said array;

4) contacting said bound analytes with said signal generating means; and 5) detecting or quantifying the presence of said analytes.

906. The process of claim 905, wherein said solid surfaces are porous or non-porous.

907. The process of claim 906, wherein said porous solid surfaces are selected from the group consisting of polyacrylamide and agarose.

908. The process of claim 906, wherein said non-porous solid surfaces comprises glass or plastic.

909. The process of claim 905, wherein said solid surfaces are transparent, translucent, opaque or reflective.

910. The process of claim 905, wherein said fixed or immobilized nucleic acid is selected from the group consisting of DNA, RNA and analogs thereof.

911. The process of claim 910, wherein said analogs comprise PNA.

912. The process of claims 910 or 911, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

913. The process of claim 905, wherein said nucleic acid portion is selected from the group consisting of DNA, RNA and analogs thereof.

914. The process of claim 913, wherein said analogs comprise PNA.

915. The process of claims 913 or 914, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

916. The process of claim 905, wherein said non-nucleic acid portions are selected from the group consisting of peptides, proteins, ligands, enzyme substrates, hormones, receptors, drugs and a combination of any of the foregoing.

917. The process of claim 905, wherein said signal generating means comprise direct signal generating means and indirect signal generating means.

918. The process of claim 917, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

919. The process of claim 917, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

920. The process of claim 919, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

921. A process for detecting or quantifying analytes of interest, said process comprising the steps of:

1) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of said discrete areas comprise nucleic acids fixed or immobilized to said discrete areas, b) chimeric compositions comprising:

i) a nucleic acid portion; and ii) a non-nucleic acid portion;

said nucleic acid portion comprising at least one sequence, wherein said non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when said non-nucleic acid portion is a peptide or protein, said nucleic acid portion does not comprises sequences which are either identical or complementary to sequences that code for said peptide or protein;

c) a sample containing or suspected of containing said analytes of interest; and d) signal generating means;

2) contacting said array with said chimeric compositions to hybridize the nucleic acid portions of said chimeric compositions to complementary nucleic acids fixed or immobilized to said array;

3) labeling said analytes of interest with said signal generating means;

4) contacting said array with the labeled analytes to bind said analytes to said non-nucleic acid portion; and 5) detecting or quantifying the presence of said analytes.

922. The process of claim 921, wherein said solid surfaces are porous or non-porous.

923. The process of claim 922, wherein said porous solid surfaces are selected from the group consisting of polyacrylamide and agarose.

924. The process of claim 922, wherein said non-porous solid surfaces comprises glass or plastic.

925. The process of claim 921, wherein said solid surfaces are transparent, translucent, opaque or reflective.

926. The process of claim 921, wherein said fixed or immobilized nucleic acid is selected from the group consisting of DNA, RNA and analogs thereof.

927. The process of claim 926, wherein said analogs comprise PNA.

928. The process of claims 926 or 927, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

929. The process of claim 921, wherein said nucleic acid portion is selected from the group consisting of DNA, RNA and analogs thereof.

930. The process of claim 929, wherein said analogs comprise PNA.

931. The process of claims 929 or 930, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

932. The process of claim 921, wherein said non-nucleic acid portions are selected from the group consisting of peptides, proteins, ligands, enzyme substrates, hormones, receptors, drugs and a combination of any of the foregoing.

933. The process of claim 921, wherein said signal generating means comprise direct signal generating means and indirect signal generating means.

934. The process of claim 933, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

935. The process of claim 933, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

936. The process of claim 935, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.

937. A process for detecting or quantifying analytes of interest, said process comprising the steps of:

1) providing a) an array of solid surfaces comprising a plurality of discrete areas; wherein at least two of said discrete areas comprise nucleic acids fixed or immobilized to said discrete areas, b) chimeric compositions comprising:

i) a nucleic acid portion; and ii) a non-nucleic acid portion;

said nucleic acid portion comprising at least one sequence, wherein said non-nucleic acid portion has a binding affinity for analytes of interest, and wherein when said non-nucleic acid portion is a peptide or protein, said nucleic acid portion does not comprises sequences which are either identical or complementary to sequences that code for said peptide or protein;

c) a sample containing or suspected of containing said analytes of interest; and d) signal generating means;

2) labeling said analytes of interest with said signal generating means;

3) contacting said chimeric compositions with the labeled analytes to bind said analytes to said non-nucleic acid portion;

4) contacting said array with said chimeric compositions to hybridize the nucleic acid portions of said chimeric compositions to complementary nucleic acids fixed or immobilized to said array; and 5) detecting or quantifying the presence of said analytes.

938. The process of claim 937, wherein said solid surfaces are porous or non-porous.

939. The process of claim 938, wherein said porous solid surfaces are selected from the group consisting of polyacrylamide and agarose.

940. The process of claim 938, wherein said non-porous solid surfaces comprises glass or plastic.

941. The process of claim 937, wherein said solid surfaces are transparent, translucent, opaque or reflective.

942. The process of claim 937, wherein said fixed or immobilized nucleic acid is selected from the group consisting of DNA, RNA and analogs thereof.

943. The process of claim 942, wherein said analogs comprise PNA.

944. The process of claims 942 or 943, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

945. The process of claim 937, wherein said nucleic acid portion is selected from the group consisting of DNA, RNA and analogs thereof.

946. The process of claim 945, wherein said analogs comprise PNA.

947. The process of claims 945 or 946, wherein said nucleic acids or analogs are modified on any one of the sugar, phosphate or base moieties.

948. The process of claim 937, wherein said non-nucleic acid portions are selected from the group consisting of peptides, proteins, ligands, enzyme substrates, hormones, receptors, drugs and a combination of any of the foregoing.

949. The process of claim 937, wherein said signal generating means comprise direct signal generating means and indirect signal generating means.

950. The process of claim 949, wherein said direct signal generation is selected from the group consisting of a fluorescent compound, a phosphorescent compound, a chemiluminescent compound, a chelating compound, an electron dense compound, a magnetic compound, an intercalating compound, an energy transfer compound and a combination of any of the foregoing.

951. The process of claim 949, wherein said indirect signal generation is selected from the group consisting of an antibody, an antigen, a hapten, a receptor, a hormone, a ligand, an enzyme and a combination of any of the foregoing.

952. The process of claim 951, wherein said enzyme catalyzes a reaction selected from the group consisting of a fluorogenic reaction, a chromogenic reaction and a chemiluminescent reaction.