US20020177141A1 - Detection of nucleic acid amplification reactions using bead arrays - Google Patents

Detection of nucleic acid amplification reactions using bead arrays Download PDF

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US20020177141A1
US20020177141A1 US10/021,906 US2190601A US2002177141A1 US 20020177141 A1 US20020177141 A1 US 20020177141A1 US 2190601 A US2190601 A US 2190601A US 2002177141 A1 US2002177141 A1 US 2002177141A1
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primer
probe
nucleic acid
enzyme
sequence
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Mark Chee
Kevin Gunderson
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Illumina Inc
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869Methods for sequencing
    • C12Q1/6874Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00497Features relating to the solid phase supports
    • B01J2219/005Beads
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00596Solid-phase processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00646Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports
    • B01J2219/00648Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports by the use of solid beads
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00603Making arrays on substantially continuous surfaces
    • B01J2219/00659Two-dimensional arrays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068Means for controlling the apparatus of the process
    • B01J2219/00702Processes involving means for analysing and characterising the products
    • B01J2219/00707Processes involving means for analysing and characterising the products separated from the reactor apparatus
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
    • B01J2219/00722Nucleotides
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/06Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00Nanotechnology
    • Y10S977/902Specified use of nanostructure
    • Y10S977/904Specified use of nanostructure for medical, immunological, body treatment, or diagnosis
    • Y10S977/924Specified use of nanostructure for medical, immunological, body treatment, or diagnosis using nanostructure as support of dna analysis

Definitions

  • the present invention is a continuation-in-part of U.S. Ser. No. 60/161,148, filed Oct. 22, 1999, which is a continuation-in-part of U.S. Ser. No. 60/135,051, filed May 20, 1999, and a continuation-in-part of U.S. Ser. No. 60/160,027, filed Oct. 22, 1999, which is a continuation-in-part of U.S. Ser. No. 60/130,089, filed Apr. 20, 1999, all of which are pending.
  • the invention relates to compositions and methods useful in the detection and quantification of a nucleic acid target using a variety of amplification techniques, including both signal amplification and target amplification. Detection proceeds through the use of a label that is associated with the amplified signal or target, either directly or indirectly, to allow optical detection of the light absorbing label using a microsphere array sensor.
  • a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4:48-51 (1993)).
  • the requirement for sensitivity i.e. low detection limits
  • PCR polymerase chain reaction
  • other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis as outlined below (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 (1993)).
  • Target amplification involves the amplification (i.e. replication) of the target sequence to be detected, resulting in a significant increase in the number of target molecules.
  • Target amplification strategies include the polymerase chain reaction (PCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA).
  • amplification strategies include the ligase chain reaction (LCR), cycling probe technology (CPT), invasive cleavage techniques such as InvaderTM technology, Q-Beta replicase (Q ⁇ R) technology, and the use of “amplification probes” such as “branched DNA” that result in multiple label probes binding to a single target sequence.
  • LCR ligase chain reaction
  • CPT cycling probe technology
  • Q ⁇ R Q-Beta replicase
  • PCR polymerase chain reaction
  • PCR there are a number of variations of PCR which also find use in the invention, including “quantitative competitive PCR” or “QC-PCR”, “arbitrarily primed PCR” or “AP-PCR”, “immuno-PCR”, “Alu-PCR”, “PCR single strand conformational polymorphism” or “PCR-SSCP”, allelic PCR (see Newton et al. Nucl. Acid Res. 17:2503 91989); “reverse transcriptase PCR” or “RT-PCR”, “biotin capture PCR”, “vectorette PCR”, “panhandle PCR”, and “PCR select cDNA subtraction”, among others.
  • SDA Strand displacement amplification
  • NASBA Nucleic acid sequence based amplification
  • Cycling probe technology is a nucleic acid detection system based on signal or probe amplification rather than target amplification, such as is done in polymerase chain reactions (PCR). Cycling probe technology relies on a molar excess of labeled probe which contains a scissile linkage of RNA. Upon hybridization of the probe to the target, the resulting hybrid contains a portion of RNA:DNA. This area of RNA:DNA duplex is recognized by RNAseH and the RNA is excised, resulting in cleavage of the probe. The probe now consists of two smaller sequences which may be released, thus leaving the target intact for repeated rounds of the reaction. The unreacted probe is removed and the label is then detected. CPT is generally described in U.S.
  • the oligonucleotide ligation assay (OLA; sometimes referred to as the ligation chain reaction (LCR)) involve the ligation of at least two smaller probes into a single long probe, using the target sequence as the template for the ligase.
  • OLA oligonucleotide ligation assay
  • LCR ligation chain reaction
  • InvaderTM technology is based on structure-specific polymerases that cleave nucleic acids in a site-specific manner. Two probes are used: an “invader” probe and a “signalling” probe, that adjacently hybridize to a target sequence with a non-complementary overlap. The enzyme cleaves at the overlap due to its recognition of the “tail”, and releases the “tail” with a label. This can then be detected.
  • the InvaderTM technology is described in U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669, all of which are hereby incorporated by reference.
  • Rolling circle amplification is based on extension of a circular probe that has hybridized to a target sequence. A polymerase is added that extends the probe sequence. As the circular probe has no terminus, the polymerase repeatedly extends the circular probe resulting in concatamers of the circular probe. As such, the probe is amplified. Rolling-circle amplification is generally described in Baner et al. (1998) Nuc. Acids Res. 26:5073-5078; Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88:189-193; and Lizardi et al. (1998) Nat Genet. 19:225-232, all of which are incorporated by reference in their entirety.
  • Branched DNA signal amplification relies on the synthesis of branched nucleic acids, containing a multiplicity of nucleic acid “arms” that function to increase the amount of label that can be put onto one probe.
  • This technology is generally described in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporated by reference.
  • dendrimers of nucleic acids serve to vastly increase the amount of label that can be added to a single molecule, using a similar idea but different compositions.
  • This technology is as described in U.S. Pat. No. 5,175,270 and Nilsen et al., J. Theor. Biol. 187:273 (1997), both of which are incorporated herein by reference.
  • the present invention provides a method for detecting a first target nucleic acid sequence.
  • the method comprises hybridizing at least a first primer nucleic acid to the first target sequence to form a first hybridization complex, contacting the first hybridization complex with a first enzyme to form a modified first primer nucleic acid, disassociating the first hybridization complex, contacting the modified first primer nucleic acid with an array comprising a substrate with a surface comprising discrete sites and a population of microspheres comprising at least a first subpopulation comprising a first capture probe such that the first capture probe and the modified primer form an assay complex, wherein the microspheres are distributed on the surface, and detecting the presence of the modified primer nucleic acid.
  • the method further comprises hybridizing at least a second primer nucleic acid to a second target sequence that is substantially complementary to the first target sequence to form a second hybridization complex, contacting the second hybridization complex with the first enzyme to form modified second primer nucleic acid, disassociating the second hybridization complex and forming a second assay complex comprising the modified second primer nucleic acid and a second capture probe on a second subpopulation.
  • the primer forms a circular probe following hybridization with the target nucleic acid to form a first hybridization complex and contacting the first hybridization complex with a first enzyme comprising a ligase such that the oligonucleotide ligation assay (OLA) occurs.
  • a first enzyme comprising a ligase such that the oligonucleotide ligation assay (OLA) occurs.
  • OLA oligonucleotide ligation assay
  • the first enzyme comprises a DNA polymerase and the modification is an extension of the primer such that the polymerase chain reaction (PCR) occurs.
  • the first enzyme comprises a ligase and the modification comprises a ligation of the first primer which hybridizes to a first domain of the first target sequence, to a third primer which hybridizes to a second adjacent domain of the first target sequence such that the ligase chain reaction (LCR) occurs.
  • LCR ligase chain reaction
  • the first primer comprises a first probe sequence, a first scissile linkage and a second probe sequence, wherein the first enzyme will cleave the scissile linkage resulting in the separation of the first and second probe sequences and the disassociation of the first hybridization complex, leaving the first target sequence intact such that the cycling probe technology (CPT) reaction occurs.
  • CPT cycling probe technology
  • the method further comprises the addition of a second enzyme comprising a nicking enzyme that nicks the extended first primer leaving the first target sequence intact, and extending from the nick using the polymerase, and thereby displacing the first newly synthesized strand and generating a second newly synthesized strand such that strand displacement amplification (SBA) occurs.
  • a second enzyme comprising a nicking enzyme that nicks the extended first primer leaving the first target sequence intact, and extending from the nick using the polymerase, and thereby displacing the first newly synthesized strand and generating a second newly synthesized strand such that strand displacement amplification (SBA) occurs.
  • SBA strand displacement amplification
  • the method further comprises the addition of a second enzyme comprising an RNA degrading enzyme that degrades the first target sequence, the addition of a third primer that hybridizes to the first newly synthesized DNA strand, the addition of a third enzyme comprising a DNA polymerase that extends the third primer to form a second newly synthesized DNA strand, to form a newly synthesized DNA hybrid, the addition of a fourth enzyme comprising an RNA polymerase that recognizes the RNA polymerase promoter and generates at least one newly synthesized RNA strand from the DNA hybrid, such that nucleic acid sequence-based amplification (NASBA) occurs.
  • NASBA nucleic acid sequence-based amplification
  • the method further comprises hybridizing a signalling primer to the target sequence, the enzyme comprises a structure-specific cleaving enzyme and the modification comprises a cleavage of said signalling primer, such that the invasive cleavage reaction occurs.
  • An additional aspect of the invention is a method for detecting a target nucleic acid sequence comprising hybridizing a first primer to a first target sequence to form a first hybridization complex, contacting the first hybridization complex with a first enzyme to extend the first primer to form a first newly synthesized strand and form a nucleic acid hybrid that comprises an RNA polymerase promoter, contacting the hybrid with an RNA polymerase that recognizes the RNA polymerase promoter and generates at least one newly synthesized RNA strand, contacting the newly synthesized RNA strand with an array comprising a substrate with a surface comprising discrete sites and a population of microspheres comprising at least a first subpopulation comprising a first capture probe; such that the first capture probe and the modified primer form an assay complex; wherein the microspheres are distributed on the surface and detecting the presence of the newly synthesized RNA strand.
  • method comprises hybridizing a second primer comprising an RNA polymerase promoter sequence to the RNA sequence to form a second hybridization complex, contacting the second hybridization complex with a second enzyme to extend the second primer to form a second newly synthesized strand and form a nucleic acid hybrid; and degrading the RNA sequence to leave the second newly synthesized strand as the first target sequence.
  • the degrading is done by the addition of an RNA degrading enzyme.
  • the degrading is done by RNA degrading activity of reverse transcriptase.
  • the method comprises hybridizing a second primer comprising an RNA polymerase promoter sequence to the DNA sequence to form a second hybridization complex, contacting the second hybridization complex with a second enzyme to extend the second primer to form a second newly synthesized strand and form a nucleic acid hybrid, and denaturing the nucleic acid hybrid such that the second newly synthesized strand is the first target sequence.
  • kits for the detection of a first target nucleic acid sequence comprises at least a first nucleic acid primer substantially complementary to at least a first domain of the target sequence, at least a first enzyme that will modify the first nucleic acid primer, and an array comprising a substrate with a surface comprising discrete sites, and a population of microspheres comprising at least a first and a second subpopulation, wherein each subpopulation comprises a bioactive agent, wherein the microspheres are distributed on the surface.
  • kits for the detection of a PCR reaction wherein the first enzyme is a thermostable DNA polymerase.
  • kits for the detection of a LCR reaction wherein the first enzyme is a ligase and the kit comprises a first nucleic acid primer substantially complementary to a first domain of the first target sequence and a third nucleic acid primer substantially complementary to a second adjacent domain of the first target sequence.
  • kits for the detection of a strand displacement amplification (SDA) reaction wherein the first enzyme is a polymerase and the kit further comprises a nicking enzyme.
  • SDA strand displacement amplification
  • kits for the detection of a NASBA reaction wherein the first enzyme is a reverse transcriptase, and the kit comprises a second enzyme comprising an RNA degrading enzyme, a third primer, a third enzyme comprising a DNA polymerase and a fourth enzyme comprising an RNA polymerase.
  • kits for the detection of an invasive cleavage reaction wherein the first enzyme is a structure-specific cleaving enzyme, and the kit comprises a signaling primer.
  • FIGS. 1A, 1B and 1 C depict three different embodiments for attaching a target sequence to an array.
  • the solid support 5 has microsphere 10 with capture probe 20 linked via a linker 15 .
  • FIG. 1A depicts direct attachment; the capture probe 20 hybridizes to a first portion of the target sequence 25 .
  • FIG. 1B depicts the use of a capture extender probe 30 that has a first portion that hybridizes to the capture probe 20 and a second portion that hybridizes to a first domain of the target sequence 25 .
  • FIG. 1C shows the use of an adapter sequence 35 , that has been added to the target sequence, for example during an amplification reaction as outlined herein.
  • FIGS. 2A and 2B depict two preferred embodiments of SBE amplification.
  • FIG. 2A shows extension primer 40 hybridized to the target sequence 25 .
  • the extension primer is modified to form a labelled primer 41 .
  • the reaction can be repeated and then the labelled primer is added to the array as above.
  • FIG. 2B depicts the same reaction but using adapter sequences.
  • FIGS. 3A and 3B depict two preferred embodiments of OLA amplification.
  • FIG. 3A depicts a first ligation probe 45 and a second ligation probe 50 with a label 55 .
  • the probes are ligated.
  • the reaction can be repeated and then the ligated primer is added to the array as above.
  • FIG. 3B depicts the same reaction but using adapter sequences.
  • FIG. 4 depicts a preferred embodiment of the invasive cleavage reaction.
  • the signaling probe 65 comprises two portions, a detection sequence 67 and a signaling portion 66 .
  • the signaling portion can serve as an adapter sequence.
  • the signaling portion generally comprises the label 55 , although as will be appreciated by those in the art, the label may be on the detection sequence as well.
  • a capture tag 60 may also be used.
  • the structure is cleaved, releasing the signaling portion 66 . The reaction can be repeated and then the signaling portion is added to the array as above.
  • FIGS. 5A and 5B depict two preferred embodiments of CPT amplification.
  • a CPT primer 70 comprising a label 55 , a first probe sequence 71 and a second probe sequence 73 , separated by a scissile linkage 72 , and optionally comprising a capture tag 60 , is hybridized to the target sequence 25 .
  • the scissile linkage is cleaved.
  • the reaction can be repeated and then the probe sequence comprising the label is added to the array as above.
  • FIG. 5B depicts the same reaction but using adapter sequences.
  • FIG. 6 depicts OLA/RCA amplification using a single “padlock probe” 57 .
  • the padlock probe is hybridized with a target sequence 25 .
  • the probe 57 is complementary to the target sequence 26 , ligation of the probe termini occurs forming a circular probe 28 .
  • the probe 57 is not complementary to the target sequence 27 , ligation does not occur.
  • Addition of polymerase and nucleotides to the circular probe results amplification of the probe 58 .
  • Cleavage of the amplified probe 58 yields fragments 59 that hybridize with an identifier probe 21 immobilized on a microsphere 10 .
  • FIG. 7 depicts an alternative method of OLA/RCA.
  • An immobilized first OLA primer 45 is hybridized with a target sequence 25 and a second OLA primer 50 .
  • the first and second OLA primers are ligated to form a ligated oligonucleotide 56 .
  • the immobilized ligated oligonucleotide is distributed on an array.
  • An RCA probe 57 and polymerase are added to the array resulting in amplification of the circular RCA probe 58 .
  • This invention is directed to the detection (and optionally quantification) of products of nucleic acid amplification reactions, using bead arrays for detection of the amplification products.
  • Suitable amplification methods include both target amplification and signal amplification and include, but are not limited to, polymerase chain reaction (PCR), ligation chain reaction (sometimes referred to as oligonucleotide ligase amplification OLA), cycling probe technology (CPT), strand displacement assay (SDA), transcription mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), and invasive cleavage technology.
  • PCR polymerase chain reaction
  • ligation chain reaction sometimes referred to as oligonucleotide ligase amplification OLA
  • CPT cycling probe technology
  • SDA strand displacement assay
  • TMA transcription mediated amplification
  • NASBA nucleic acid sequence based amplification
  • RCA rolling circle amplification
  • PCR generally requires two primers, dNTPs and a DNA polymerase; LCR requires two primers that adjacently hybridize to the target sequence and a ligase; CPT requires one cleavable primer and a cleaving enzyme; invasive cleavage requires two primers and a cleavage enzyme; etc.
  • a target nucleic acid is added to a reaction mixture that comprises the necessary amplification components, and a modified primer is formed.
  • the modified primer comprises a detectable label, such as a fluorescent label, which is either incorporated by the enzyme or present on the original primer.
  • a detectable label such as a fluorescent label
  • the unreacted primers are removed, in a variety of ways, as will be appreciated by those in the art and outlined herein.
  • the hybridization complex is then disassociated, and the modified primer is detected and optionally quantitated by a microsphere array.
  • the newly modified primer serves as a target sequence for a secondary reaction, which then produces a number of amplified strands, which can be detected as outlined herein.
  • the present invention provides compositions and methods for detecting the presence or absence of target nucleic acid sequences in a sample.
  • the sample solution may comprise any number of things, including, but not limited to, bodily fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen, of virtually any organism, with mammalian samples being preferred and human samples being particularly preferred); environmental samples (including, but not limited to, air, agricultural, water and soil samples); biological warfare agent samples; research samples; purified samples, such as purified genomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc.; As will be appreciated by those in the art, virtually any experimental manipulation may have been done on the sample.
  • nucleic acid or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together.
  • a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sblul et al., Eur. J.
  • nucleic acid analogs may find use in the present invention.
  • mixtures of naturally occurring nucleic acids and analogs can be made.
  • mixtures of different nucleic acid analogs, and mixtures of naturally occuring nucleic acids and analogs may be made.
  • PNA peptide nucleic acids
  • These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages.
  • the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9° C. This allows for better detection of mismatches.
  • Tm melting temperature
  • RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch.
  • the non-ionic PNA backbone the drop is closer to 7-9° C. This allows for better detection of mismatches.
  • hybridization of the bases attached to these backbones is relatively insensitive to salt concentration.
  • the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
  • nucleoside includes nucleotides as well as nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides.
  • nucleoside includes non-naturally occuring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
  • compositions and methods of the invention are directed to the detection of target sequences.
  • target sequence or “target nucleic acid” or grammatical equivalents herein means a nucleic acid sequence on a single strand of nucleic acid.
  • the target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others.
  • the target sequence may be a target sequence from a sample, or a secondary target such as a product of a reaction such as a detection sequence from an invasive cleavage reaction, a ligated probe from an OLA reaction, an extended probe from a PCR reaction, etc.
  • a target sequence from a sample is amplified to produce a secondary target that is detected, as outlined herein.
  • an amplification step is done using a signal probe that is amplified, again producing a secondary target that is detected.
  • the target sequence may be any length, with the understanding that longer sequences are more specific.
  • the complementary target sequence may take many forms. For example, it may be contained within a larger nucleic acid sequence, i.e. all or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, among others.
  • probes are made to hybridize to target sequences to determine the presence or absence of the target sequence in a sample.
  • the target sequence may also be comprised of different target domains; for example, in “sandwich” type assays as outlined below, a first target domain of the sample target sequence may hybridize to a capture probe or a portion of capture extender probe, a second target domain may hybridize to a portion of an amplifier probe, a label probe, or a different capture or capture extender probe, etc.
  • the target domains may be adjacent (i.e. contiguous) or separated.
  • a first primer may hybridize to a first target domain and a second primer may hybridize to a second target domain; either the domains are adjacent, or they may be separated by one or more nucleotides, coupled with the use of a polymerase and dNTPs, as is more fully outlined below.
  • first and second are not meant to confer an orientation of the sequences with respect to the 5′-3′ orientation of the target sequence. For example, assuming a 5′-3′ orientation of the complementary target sequence, the first target domain may be located either 5′ to the second domain, or 3′ to the second domain.
  • the target sequence is prepared using known techniques.
  • the sample may be treated to lyse the cells, using known lysis buffers, sonication, electroporation, etc., with purification occuring as needed, as will be appreciated by those in the art.
  • the reactions outlined herein may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below.
  • the reaction may include a variety of other reagents which may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g.
  • albumin which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions.
  • reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target.
  • double stranded target nucleic acids are denatured to render them single stranded so as to permit hybridization of the primers and other probes of the invention.
  • a preferred embodiment utilizes a thermal step, generally by raising the temperature of the reaction to about 95° C., although pH changes and other techniques may also be used.
  • a primer nucleic acid is then contacted to the target sequence to form a hybridization complex.
  • primer nucleic acid herein is meant a probe nucleic acid that will hybridize to some portion, i.e. a domain, of the target sequence.
  • Probes of the present invention are designed to be complementary to a target sequence (either the target sequence of the sample or to other probe sequences, as is described below), such that hybridization of the target sequence and the probes of the present invention occurs. As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention.
  • the sequence is not a complementary target sequence.
  • substantially complementary herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions.
  • hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al, hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993).
  • stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g.
  • Stringent conditions may also be achieved with the addition of helix destabilizing agents such as formamide.
  • the hybridization conditions may also vary when a non-ionic backbone, i.e. PNA is used, as is known in the art.
  • cross-linking agents may be added after target binding to cross-link, i.e. covalently attach, the two strands of the hybridization complex.
  • the assays are generally run under stringency conditions which allows formation of the hybridization complex only in the presence of target.
  • Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent concentration, etc.
  • the size of the primer nucleic acid may vary, as will be appreciated by those in the art, in general varying from 5 to 500 nucleotides in length, with primers of between 10 and 100 being preferred, between 15 and 50 being particularly preferred, and from 10 to 35 being especially preferred, depending on the use and amplification technique.
  • the different amplification techniques may have further requirements of the primers, as is more fully described below.
  • an enzyme sometimes termed an “amplification enzyme” is used to modify the primer.
  • the enzymes may be added at any point during the assay, either prior to, during, or after the addition of the primers.
  • the identity of the enzyme will depend on the amplification technique used, as is more fully outlined below.
  • the modification will depend on the amplification technique, as outlined below.
  • the hybridization complex is disassociated.
  • dissociation is by modification of the assay conditions.
  • the modified primer no longer hybridizes to the target nucleic acid and dissociates. Either one or both of these aspects can be employed in signal and target amplification reactions as described below.
  • the amplification steps are repeated for a period of time to allow a number of cycles, depending on the number of copies of the original target sequence and the sensitivity of detection, with cycles ranging from 1 to thousands, with from 10 to 100 cycles being preferred and from 20 to 50 cycles being especially preferred.
  • the modified primer comprises a detectable label, such as a fluorescent label, which is either incorporated by the enzyme or present on the original primer, and the modified primer is added to a microsphere array such is generally described in U.S. Ser. Nos.
  • microsphere array comprises subpopulations of microspheres that comprise capture probes that will hybridize to the modified primers. Detection proceeds via detection of the label as an indication of the presence, absence or amount of the target sequence, as is more fully outlined below.
  • the amplification is target amplification.
  • Target amplification involves the amplification (replication) of the target sequence to be detected, such that the number of copies of the target sequence is increased.
  • Suitable target amplification techniques include, but are not limited to, the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA) and nucleic acid sequence based amplification (NASBA).
  • the target amplification technique is PCR.
  • the polymerase chain reaction (PCR) is widely used and described, and involves the use of primer extension combined with thermal cycling to amplify a target sequence; see U.S. Pat. Nos. 4,683,195 and 4,683,202, and PCR Essential Data, J. W. Wiley & sons, Ed. C. R. Newton, 1995, all of which are incorporated by reference.
  • PCR there are a number of variations of PCR which also find use in the invention, including “quantitative competitive PCR” or “QC-PCR”, “arbitrarily primed PCR” or “AP-PCR”, “immuno-PCR”, “Alu-PCR”, “PCR single strand conformational polymorphism” or “PCR-SSCP”, “reverse transcriptase PCR” or “RT-PCR”, “biotin capture PCR”, “vectorette PCR”, “panhandle PCR”, and “PCR select cDNA subtraction”, “allele-specific PCR”, among others.
  • PCR may be briefly described as follows.
  • a double stranded target nucleic acid is denatured, generally by raising the temperature, and then cooled in the presence of an excess of a PCR primer, which then hybridizes to the first target strand.
  • a DNA polymerase then acts to extend the primer with dNTPs, resulting in the synthesis of a new strand forming a hybridization complex.
  • the sample is then heated again, to disassociate the hybridization complex, and the process is repeated.
  • a second PCR primer for the complementary target strand, rapid and exponential amplification occurs.
  • PCR steps are denaturation, annealing and extension.
  • the particulars of PCR are well known, and include the use of a thermostable polymerase such as Taq I polymerase and thermal cycling.
  • the PCR reaction requires at least one PCR primer, a polymerase, and a set of dNTPs.
  • the primers may comprise the label, or one or more of the dNTPs may comprise a label.
  • the capture probes on the beads of the array are designed to be substantially complementary to the extended part of the primer; that is, unextended primers will not bind to the capture probes.
  • unreacted probes may be removed prior to addition to the array.
  • the target amplification technique is SDA.
  • Strand displacement amplification (SDA) is generally described in Walker et al., in Molecular Methods for Virus Detection, Academic Press, Inc., 1995, and U.S. Pat. Nos. 5,455,166 and 5,130,238, all of which are hereby expressly incorporated by reference in their entirety.
  • SDA may be described as follows.
  • a single stranded target nucleic acid is contacted with an SDA primer.
  • An “SDA primer” generally has a length of 25-100 nucleotides, with SDA primers of approximately 35 nucleotides being preferred.
  • An SDA primer is substantially complementary to a region at the 3′ end of the target sequence, and the primer has a sequence at its 5′ end (outside of the region that is complementary to the target) that is a recognition sequence for a restriction endonuclease, sometimes referred to herein as a “nicking enzyme” or a “nicking endonuclease”, as outlined below.
  • the SDA primer then hybridizes to the target sequence.
  • the SDA reaction mixture also contains a polymerase (an “SDA polymerase”, as outlined below) and a mixture of all four deoxynucleoside-triphosphates (also called deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP), at least one species of which is a substituted or modified dNTP; thus, the SDA primer is modified, i.e. extended, to form a modified primer, sometimes referred to herein as a “newly synthesized strand”.
  • the substituted dNTP is modified such that it will inhibit cleavage in the strand containing the substituted dNTP but will not inhibit cleavage on the other strand.
  • Suitable substituted dNTPs include, but are not limited, 2′deoxyadenosine 5′-O-(1-thiotriphosphate), 5-methyideoxycytidine 5′-triphosphate, 2′-deoxyuridine 5′-triphosphate, adn 7-deaza-2′-deoxyguanosine 5′-triphosphate.
  • the substitution of the dNTP may occur after incorporation into a newly synthesized strand; for example, a methylase may be used to add methyl groups to the synthesized strand.
  • the polymerase may have 5′ ⁇ 3′ exonuclease activity. However, if less than all the nucleotides are substituted, the polymerase preferably lacks 5′ ⁇ 3′ exonuclease activity.
  • the recognition site/endonuclease pair can be any of a wide variety of known combinations.
  • the endonuclease is chosen to cleave a strand either at the recognition site, or either 3′ or 5′ to it, without cleaving the complementary sequence, either because the enzyme only cleaves one strand or because of the incorporation of the substituted nucleotides.
  • Suitable recognition site/endonuclease pairs are well known in the art; suitable endonucleases include, but are not limited to, HincII, HindII, AvaI, Fnu4HI, TthIIII, NcII, BstXI, BamHI, etc.
  • a chart depicting suitable enzymes, and their corresponding recognition sites and the modified dNTP to use is found in U.S. Pat. No. 5,455,166, hereby expressly incorporated by reference.
  • a polymerase (an “SDA polymerase”) is used to extend the newly nicked strand, 5′ ⁇ 3 ′, thereby creating another newly synthesized strand.
  • the polymerase chosen should be able to intiate 5′ ⁇ 3′ polymerization at a nick site, should also displace the polymerized strand downstream from the nick, and should lack 5′ ⁇ 3′ exonuclease activity (this may be additionally accomplished by the addition of a blocking agent).
  • suitable polymerases in SDA include, but are not limited to, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29 DNA polymerase.
  • the SDA reaction requires, in no particular order, an SDA primer, an SDA polymerase, a nicking endonuclease, and dNTPs, at least one species of which is modified.
  • an SDA primer an SDA polymerase
  • a nicking endonuclease a nicking endonuclease
  • dNTPs a species of which is modified.
  • preferred embodiments utilize capture probes complementary to the newly synthesized portion of the primer, rather than the primer region, to allow unextended primers to be removed.
  • SDA does not require thermocycling.
  • the temperature of the reaction is generally set to be high enough to prevent non-specific hybridization but low enough to allow specific hybridization; this is generally from about 37° C. to about 42° C., depending on the enzymes.
  • a second amplification reaction can be done using the complementary target sequence, resulting in a substantial increase in amplification during a set period of time. That is, a second primer nucleic acid is hybridized to a second target sequence, that is substantially complementary to the first target sequence, to form a second hybridization complex. The addition of the enzyme, followed by disassociation of the second hybridization complex, results in the generation of a number of newly synthesized second strands.
  • NASBA Nucleic Acid Sequence Based Amplification
  • TMA Transcription Mediated Amplification
  • the target amplification technique is nucleic acid sequence based amplification (NASBA).
  • NASBA is generally described in U.S. Pat. No. 5,409,818; Sooknanan et al., Nucleic Acid Sequence-Based Amplification, Ch. 12 (pp. 261-285) of Molecular Methods for Virus Detection, Academic Press, 1995; and “Profiting from Gene-based Diagnostics”, CTB International Publishing Inc., N.J., 1996, all of which are incorporated by reference.
  • NASBA is very similar to both TMA and QBR.
  • Transcription mediated amplification (TMA) is generally described in U.S. Pat. Nos.
  • NASBA utilizes the addition of RNAse H to effect RNA degradation
  • TMA relies on inherent RNAse H activity of the reverse transcriptase.
  • a single stranded target nucleic acid is contacted with a first primer, generally referred to herein as a “NASBA primer” (although “TMA primer” is also suitable).
  • a first primer generally referred to herein as a “NASBA primer” (although “TMA primer” is also suitable).
  • NASBA primer a first primer
  • TMA primer TMA primer
  • the first primer is preferably a DNA primer that has at its 3′ end a sequence that is substantially complementary to the 3′ end of the first template.
  • the first primer also has an RNA polymerase promoter at its 5′ end (or its complement (antisense), depending on the configuration of the system).
  • the first primer is then hybridized to the first template to form a first hybridization complex.
  • the reaction mixture also includes a reverse transcriptase enzyme (an “NASBA reverse transcriptase”) and a mixture of the four dNTPs, such that the first NASBA primer is modified, i.e. extended, to form a modified first primer, comprising a hybridization complex of RNA (the first template) and DNA (the newly synthesized strand).
  • RNA-directed DNA polymerase an enzyme capable of synthesizing DNA from a DNA primer and an RNA template.
  • Suitable RNA-directed DNA polymerases include, but are not limited to, avian myloblastosis virus reverse transcriptase (“AMV RT”) and the Moloney murine leukemia virus RT.
  • AMV RT avian myloblastosis virus reverse transcriptase
  • Moloney murine leukemia virus RT Moloney murine leukemia virus RT.
  • the reverse transcriptase enzyme further comprises a RNA degrading activity as outlined below.
  • the NASBA reaction also includes an RNA degrading enzyme, also sometimes referred to herein as a ribonuclease, that will hydrolyze RNA of an RNA:DNA hybrid without hydrolyzing single- or double-stranded RNA or DNA.
  • RNA degrading enzyme also sometimes referred to herein as a ribonuclease
  • Suitable ribonucleases include, but are not limited to, RNase H from E. coli and calf thymus.
  • the ribonuclease activity degrades the first RNA template in the hybridization complex, resulting in a disassociation of the hybridization complex leaving a first single stranded newly synthesized DNA strand, sometimes referred to herein as “the second template”.
  • the NASBA reaction also includes a second NASBA primer, generally comprising DNA (although as for all the probes herein, including primers, nucleic acid analogs may also be used).
  • This second NASBA primer has a sequence at its 3′ end that is substantially complementary to the 3′ end of the second template, and also contains an antisense sequence for a functional promoter and the antisense sequence of a transcription initiation site.
  • this primer sequence when used as a template for synthesis of the third DNA template, contains sufficient information to allow specific and efficient binding of an RNA polymerase and initiation of transcription at the desired site.
  • Preferred embodiments utilizes the antisense promoter and transcription initiation site are that of the T7 RNA polymerase, although other RNA polymerase promoters and initiation sites can be used as well, as outlined below.
  • the second primer hybridizes to the second template, and a DNA polymerase, also termed a “DNA-directed DNA polymerase”, also present in the reaction, synthesizes a third template (a second newly synthesized DNA strand), resulting in second hybridization complex comprising two newly synthesized DNA strands.
  • a DNA polymerase also termed a “DNA-directed DNA polymerase”
  • RNA polymerase ribonucleoside triphosphates
  • NTPs ribonucleotides
  • the RNA polymerase sometimes referred to herein as a “DNA-directed RNA polymerase”, recognizes the promoter and specifically initiates RNA synthesis at the initiation site.
  • the RNA polymerase preferably synthesizes several copies of RNA per DNA duplex.
  • RNA polymerases include, but are not limited to, T7 RNA polymerase, and other bacteriophage RNA polymerases including those of phage T3, phage ⁇ II, Salmonella phage sp6, or Pseudomonase phage gh-1.
  • TMA and NASBA are used with starting DNA target sequences.
  • the NASBA reaction requires, in no particular order, a first NASBA primer, a second NASBA primer comprising an antisense sequence of an RNA polymerase promoter, an RNA polymerase that recognizes the promoter, a reverse transcriptase, a DNA polymerase, an RNA degrading enzyme, NTPs and dNTPs, in addition to the detection components outlined below.
  • the TMA reaction requires, in no particular order, a first TMA primer, a second TMA primer comprising an antisense sequence of an RNA polymerase promoter, an RNA polymerase that recognizes the promoter, a reverse transcriptase with RNA degrading activity, a DNA polymerase, NTPs and dNTPs, in addition to the detection components outlined below.
  • the detection of the newly synthesized strands can proceed in several ways. Direct detection can be done when the newly synthesized strands comprise detectable labels, either by incorporation into the primers or by incorporation of modified labelled nucleotides into the growing strand. Alternatively, as is more fully outlined below, indirect detection of unlabelled strands (which now serve as “targets” in the detection mode) can occur using a variety of sandwich assay configurations. As will be appreciated by those in the art, any of the newly synthesized strands can serve as the “target” for form an assay complex on a surface with a capture probe. In NASBA and TMA, it is preferable to utilize the newly formed RNA strands as the target, as this is where significant amplification occurs.
  • the amplification technique is signal amplification.
  • Signal amplification involves the use of limited number of target molecules as templates to either generate multiple signalling probes or allow the use of multiple signalling probes.
  • Signal amplification strategies include LCR, CPT, Q ⁇ R, invasive cleavage technology, and the use of amplification probes in sandwich assays.
  • SBE single base extension
  • a polymerase generally a DNA polymerase
  • a polymerase is used to extend the 3′ end of the primer with a nucleotide analog labeled a detection label as described herein.
  • nucleotide Based on the fidelity of the enzyme, a nucleotide is only incorporated into the extension primer if it is complementary to the adjacent base in the target strand. Generally, the nucleotide is derivatized such that no further extensions can occur, so only a single nucleotide is added. However, for amplification reactions, this may not be necessary.
  • detection of the label proceeds as outlined herein. See generally Sylvanen et al., Genomics 8:684-692 (1990); U.S. Pat. Nos. 5,846,710 and 5,888,819; Pastinen et al., Genomics Res. 7(6):606-614 (1997); all of which are expressly incorporated herein by reference.
  • the reaction is initiated by introducing the assay complex comprising the target sequence (i.e. the array) to a solution comprising a first nucleotide, frequently an nucleotide analog.
  • nucleoide analog in this context herein is meant a deoxynucleoside-triphosphate (also called deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP), that is further derivatized to be chain terminating.
  • dNTPs also called deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP
  • any number of nucleotide analogs may be used, as long as a polymerase enzyme will still incorporate the nucleotide at the interrogation position.
  • Preferred embodiments utilize dideoxy-triphosphate nucleotides (ddNTPs).
  • ddNTPs dideoxy-triphosphate nucleotides
  • a set of nucleotides comprising ddATP, ddCTP, ddGTP and ddTTP is used, at least one of which includes a label, and preferably all four.
  • the labels may all be the same; alternatively, different labels may be used.
  • the nucleotide analogs comprise a detectable label, which can be either a primary or secondary detectable label.
  • Preferred primary labels are those outlined above.
  • the enzymatic incorporation of nucleotides comprising fluorophores is poor under many conditions; accordingly, preferred embodiments utilize secondary detectable labels.
  • the use of secondary labels may also facilitate the removal of unextended probes.
  • the solution also comprises an extension enzyme, generally a DNA polymerase.
  • Suitable DNA polymerases include, but are not limited to, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29 DNA polymerase. If the NTP is complementary to the base of the detection position of the target sequence, which is adjacent to the extension primer, the extension enzyme will add it to the extension primer. Thus, the extension primer is modified, i.e. extended, to form a modified primer, sometimes referred to herein as a “newly synthesized strand”.
  • a limitation of this method is that unless the target nucleic acid is in sufficient concentration, the amount of unextended primer in the reaction greatly exceeds the resultant extended-labeled primer. The excess of unextended primer competes with the detection of the labeled primer in the assays described herein. Accordingly, when SBE is used, preferred embodiments utilize methods for the removal of unextended primers as outlined herein.
  • thermocycling minisequencing in which repeated cycles of annealing, primer extension, and heat denaturation using a thermocycler and thermo-stable polymerase allows the amplification of the extension probe which results in the accumulation of extended primers. For example, if the original unextended primer to target nucleic acid concentration is 100:1 and 100 thermocycles and extensions are performed, a majority of the primer will be extended.
  • the configuration of the SBE system can take on several forms.
  • the reaction may be done in solution, and then the newly synthesized strands, with the base-specific detectable labels, can be detected. For example, they can be directly hybridized to capture probes that are complementary to the extension primers, and the presence of the label is then detected.
  • the SBE reaction can occur on a surface.
  • a target nucleic acid may be captured using a first capture probe that hybridizes to a first target domain of the target, and the reaction can proceed at a second target domain.
  • the extended labeled primers are then bound to a second capture probe and detected.
  • the SBE reaction requires, in no particular order, an extension primer, a polymerase and dNTPs, at least one of which is labeled.
  • the signal amplification technique is OLA.
  • OLA which is referred to as the ligation chain reaction (LCR) when two-stranded substrates are used, involves the ligation of two smaller probes into a single long probe, using the target sequence as the template.
  • LCR the ligation chain reaction
  • the ligated probe product becomes the predominant template as the reaction progresses.
  • the method can be run in two different ways; in a first embodiment, only one strand of a target sequence is used as a template for ligation; alternatively, both strands may be used. See generally U.S. Pat. Nos.
  • the single-stranded target sequence comprises a first target domain and a second target domain, which are adjacent and contiguous.
  • a first OLA primer and a second OLA primer nucleic acids are added, that are substantially complementary to their respective target domain and thus will hybridize to the target domains.
  • These target domains may be directly adjacent, i.e. contiguous, or separated by a number of nucleotides. If they are non-contiguous, nucleotides are added along with means to join nucleotides, such as a polymerase, that will add the nucleotides to one of the primers.
  • the two OLA primers are then covalently attached, for example using a ligase enzyme such as is known in the art, to form a modified primer.
  • This forms a first hybridization complex comprising the ligated probe and the target sequence.
  • This hybridization complex is then denatured (disassociated), and the process is repeated to generate a pool of ligated probes.
  • OLA is done for two strands of a double-stranded target sequence.
  • the target sequence is denatured, and two sets of probes are added: one set as outlined above for one strand of the target, and a separate set (i.e. third and fourth primer probe nucleic acids) for the other strand of the target.
  • the first and third probes will hybridize, and the second and fourth probes will hybridize, such that amplification can occur. That is, when the first and second probes have been attached, the ligated probe can now be used as a template, in addition to the second target sequence, for the attachment of the third and fourth probes. Similarly, the ligated third and fourth probes will serve as a template for the attachment of the first and second probes, in addition to the first target strand. In this way, an exponential, rather than just a linear, amplification can occur.
  • the ligation product can be detected in a variety of ways.
  • the ligation reaction is run in solution.
  • only one of the primers carries a detectable label, e.g. the first ligation probe, and the capture probe on the bead is substantially complementary to the other probe, e.g. the second ligation probe.
  • the ligation product is detected by solid-phase oligonucleotide probes.
  • the solid-phase probes are preferably complementary to at least a portion of the ligation product.
  • the solid-phase probe is complementary to the 5′ detection oligonucleotide portion of the ligation product. This substantially reduces or eliminates false signal generated by the optically-labeled 3′ primers.
  • detection is accomplished by removing the unligated 5′ detection oligonucleotide from the reaction before application to a capture probe.
  • the unligated 5′ detection oligonucleotides are removed by digesting 3′ non-protected oligonucleotides with a 3′ exonuclease, such as, exonuclease I.
  • the ligation products are protected from exo I digestion by including, for example, 4-phosphorothioate residues at their 3′ terminus, thereby, rendering them resistant to exonuclease digestion.
  • the unligated detection oligonucleotides are not protected and are digested.
  • the target nucleic acid is immobilized on a solid-phase surface.
  • the ligation assay is performed and unligated oligonucleotides are removed by washing under appropriate stringency to remove unligated oligonucleotides.
  • the ligated oligonucleotides are eluted from the target nucleic acid using denaturing conditions, such as, 0.1 N NaOH, and detected as described herein.
  • the detection of the LCR reaction can also occur directly, in the case where one or both of the primers comprises at least one detectable label, or indirectly, using sandwich assays, through the use of additional probes; that is, the ligated probes can serve as target sequences, and detection may utilize amplification probes, capture probes, capture extender probes, label probes, and label extender probes, etc.
  • the signal amplification technique is RCA.
  • Rolling-circle amplification is generally described in Baner et al. (1998) Nuc. Acids Res. 26:5073-5078; Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88:189-193; and Lizardi et al. (1998) Nat. Genet. 19:225-232, all of which are incorporated by reference in their entirety.
  • RCA may be described in two ways. First, as is outlined in more detail below, a single probe is hybridized with a target nucleic acid. Each terminus of the probe hybridizes adjacently on the target nucleic acid and the OLA assay as described above occurs. When ligated, the probe is circularized while hybridized to the target nucleic acid. Addition of a polymerase results in extension of the circular probe. However, since the probe has no terminus, the polymerase continues to extend the probe repeatedly. Thus results in amplification of the circular probe.
  • a second alternative approach involves OLA followed by RCA.
  • an immobilized primer is contacted with a target nucleic acid. Complementary sequences will hybridize with each other resulting in an immobilized duplex.
  • a second primer is contacted with the target nucleic acid. The second primer hybridizes to the target nucleic acid adjacent to the first primer.
  • An OLA assay is performed as described above. Ligation only occurs if the primer are complementary to the target nucleic acid. When a mismatch occurs, particularly at one of the nucleotides to be ligated, ligation will not occur.
  • the ligated, immobilized, oligonucleotide is then hybridized with an RCA probe. This is a circular probe that is designed to specifically hybridize with the ligated oligonucleotide and will only hybridize with an oligonucleotide that has undergone ligation.
  • RCA is then performed as is outlined in more detail below.
  • a single oligonucleotide is used both for OLA and as the circular template for RCA (referred to herein as a “padlock probe” or a “RCA probe”). That is, each terminus of the oligonucleotide contains sequence complementary to the target nucleic acid and functions as an OLA primer as described above. That is, the first end of the RCA probe is substantially complementary to a first target domain, and the second end of the RCA probe is substantially complementary to a second target domain, adjacent to the first domain. Hybridization of the oligonucleotide to the target nucleic acid results in the formation of a hybridization complex.
  • Ligation of the “primers” results in the formation of a modified hybridization complex containing a circular probe i.e. an RCA template complex. That is, the oligonucleotide is circularized while still hybridized with the target nucleic acid. This serves as a circular template for RCA.
  • Addition of a polymerase to the RCA template complex results in the formation of an amplified product nucleic acid. Following RCA, the amplified product nucleic acid is detected (FIG. 6).
  • the polymerase may incorporate labelled nucleotides, or alternatively, a label probe is used that is substantially complementary to a portion of the RCA probe and comprises at least one label is used.
  • the polymerase can be any polymerase, but is preferably one lacking 3′ exonuclease activity (3′ exo ⁇ ).
  • suitable polymerase include but are not limited to exonuclease minus DNA Polymerase I large (Klenow) Fragment, Phi29 DNA polymerase, Taq DNA Polymerase and the like.
  • a polymerase that will replicate single-stranded DNA i.e. without a primer forming a double stranded section
  • the RCA probe contains an adapter sequence as outlined herein, with adapter capture probes on the array, for example on a microsphere when microsphere arrays are being used.
  • adapter capture probes on the array, for example on a microsphere when microsphere arrays are being used.
  • unique portions of the RCA probes for example all or part of the sequence corresponding to the target sequence, can be used to bind to a capture probe.
  • the padlock probe contains a restriction site.
  • the restriction endonuclease site allows for cleavage of the long concatamers that are typically the result of RCA into smaller individual units that hybridize either more efficiently or faster to surface bound capture probes.
  • the product nucleic acid is contacted with the appropriate restriction endonuclease. This results in cleavage of the product nucleic acid into smaller fragments.
  • the fragments are then hybridized with the capture probe that is immobilized resulting in a concentration of product fragments onto the microsphere.
  • these fragments can be detected in one of two ways: either labelled nucleotides are incorporated during the replication step, or an additional label probe is added.
  • the padlock probe comprises a label sequence; i.e. a sequence that can be used to bind label probes and is substantially complementary to a label probe.
  • a label sequence i.e. a sequence that can be used to bind label probes and is substantially complementary to a label probe.
  • the padlock probe also contains a priming site for priming the RCA reaction. That is, each padlock probe comprises a sequence to which a primer nucleic acid hybridizes forming a template for the polymerase.
  • the primer can be found in any portion of the circular probe. In a preferred embodiment, the primer is located at a discrete site in the probe. In this embodiment, the primer site in each distinct padlock probe is identical, although this is not required. Advantages of using primer sites with identical sequences include the ability to use only a single primer oligonucleotide to prime the RCA assay with a plurality of different hybridization complexes. That is, the padlock probe hybridizes uniquely to the target nucleic acid to which it is designed. A single primer hybridizes to all of the unique hybridization complexes forming a priming site for the polymerase. RCA then proceeds from an identical locus within each unique padlock probe of the hybridization complexes.
  • the primer site can overlap, encompass, or reside within any of the above-described elements of the padlock probe. That is, the primer can be found, for example, overlapping or within the restriction site or the identifier sequence. In this embodiment, it is necessary that the primer nucleic acid is designed to base pair with the chosen primer site.
  • the padlock probe of the invention contains at each terminus, sequences corresponding to OLA primers.
  • the intervening sequence of the padlock probe contain in no particular order, an adapter sequence and a restriction endonuclease site.
  • the padlock probe contains a RCA priming site.
  • the OLA/RCA is performed in solution followed by restriction endonuclease cleavage of the RCA product.
  • the cleaved product is then applied to an array comprising beads, each bead comprising a probe complementary to the adapter sequence located in the padlock probe.
  • the amplified adapter sequence correlates with a particular target nucleic acid.
  • the unique adapter sequence in each rolling circle padlock probe sequence allows diverse sets of nucleic acid sequences to be analyzed in parallel on an array, since each sequence is resolved on the basis of hybridization specificity.
  • one of the OLA primers is immobilized on the microsphere; the second primer is added in solution. Both primers hybridize with the target nucleic acid forming a hybridization complex as described above for the OLA assay.
  • the microsphere is distributed on an array.
  • a plurality of microspheres each with a unique OLA primer is distributed on the array.
  • a segment of circular DNA is hybridized to the bead-based ligated oligonucleotide forming a modified hybridization complex.
  • Addition of an appropriate polymerase (3′ exo ⁇ ), as is known in the art, and corresponding reaction buffer to the array leads to amplification of the circular DNA. Since there is no terminus to the circular DNA, the polymerase continues to travel around the circular template generating extension product until it detaches from the template. Thus, a polymerase with high processivity can create several hundred or thousand copies of the circular template with all the copies linked in one contiguous strand.
  • these copies are subsequently detected by one of two methods; either hybridizing a labeled oligo complementary to the circular target or via the incorporation of labeled nucleotides in the amplification reaction.
  • the label is detected using conventional label detection methods as described herein.
  • the circular DNA when the circular DNA contains sequences complementary to the ligated oligonucleotide it is preferable to remove the target DNA prior to contacting the ligated oligonucleotide with the circular DNA (See FIG. 7). This is done by denaturing the double-stranded DNA by methods known in the art. In an alternative embodiment, the double stranded DNA is not denatured prior to contacting the circular DNA.
  • the circular DNA when the circular DNA contains sequences complementary to the target nucleic acid, it is preferable that the circular DNA is complementary at a site distinct from the site bound to the ligated oligonucleotide. In this embodiment it is preferred that the duplex between the ligated oligonucleotide and target nucleic acid is not denatured or disrupted prior to the addition of the circular DNA so that the target DNA remains immobilized to the bead.
  • Hybridization and washing conditions are well known in the art; various degrees of stringency can be used. In some embodiments it is not necessary to use stringent hybridization or washing conditions as only microspheres containing the ligated probes will effectively hybridize with the circular DNA; microspheres bound to DNA that did not undergo ligation (those without the appropriate target nucleic acid) will not hybridize as strongly with the circular DNA as those primers that were ligated. Thus, hybridization and/or washing conditions are used that discriminate between binding of the circular DNA to the ligated primer and the unligated primer.
  • the circular probe when the circular probe is designed to hybridize to the target nucleic acid at a site distinct from the site bound to the ligated oligonucleotide, hybridization and washing conditions are used to remove or dissociate the target nucleic acid from unligated oligonucleotides while target nucleic acid hybridizing with the ligated oligonucleotides will remain bound to the beads.
  • the circular probe only hybridizes to the target nucleic acid when the target nucleic acid is hybridized with a ligated oligonucleotide that is immobilized on a bead.
  • an appropriate polymerase (3′ exo ⁇ ) is added to the array.
  • the polymerase extends the sequence of a single-stranded DNA using double-stranded DNA as a primer site.
  • the circular DNA that has hybridized with the appropriate OLA reaction product serves as the primer for the polymerase.
  • the polymerase will extend the sequence of the primer using the single-stranded circular DNA as a template. As there is no terminus of the circular DNA, the polymerase will continue to extend the sequence of the circular DNA.
  • the RCA probe comprises a discrete primer site located within the circular probe. Hybridization of primer nucleic acids to this primer site forms the polymerase template allowing RCA to proceed.
  • the polymerase creates more than 100 copies of the circular DNA. In more preferred embodiments the polymerase creates more than 1000 copies of the circular DNA; while in a most preferred embodiment the polymerase creates more than 10,000 copies or more than 50,000 copies of the template.
  • the amplified circular DNA sequence is then detected by methods known in the art and as described herein. Detection is accomplished by hybridizing with a labeled probe.
  • the probe is labeled directly or indirectly.
  • labeled nucleotides are incorporated into the amplified circular DNA product.
  • the nucleotides can be labeled directly, or indirectly as is further described herein.
  • the RCA as described herein finds use in allowing highly specific and highly sensitive detection of nucleic acid target sequences.
  • the method finds use in improving the multiplexing ability of DNA arrays and eliminating costly sample or target preparation.
  • a substantial savings in cost can be realized by directly analyzing genomic DNA on an array, rather than employing an intermediate PCR amplification step.
  • the method finds use in examining genomic DNA and other samples including mRNA.
  • the RCA finds use in allowing rolling circle amplification products to be easily detected by hybridization to probes in a solid-phase format (e.g. an array of beads).
  • An additional advantage of the RCA is that it provides the capability of multiplex analysis so that large numbers of sequences can be analyzed in parallel. By combining the sensitivity of RCA and parallel detection on arrays, many sequences can be analyzed directly from genomic DNA.
  • LCR utilizes a “chemical ligation” of sorts, as is generally outlined in U.S. Pat. Nos. 5,616,464 and 5,767,259, both of which are hereby expressly incorporated by reference in their entirety.
  • a pair of primers are utilized, wherein the first primer is substantially complementary to a first domain of the target and the second primer is substantially complementary to an adjacent second domain of the target (although, as for enzymatic ligation, if a “gap” exists, a polymerase and dNTPs may be added to “fill in” the gap).
  • Each primer has a portion that acts as a “side chain” that does not bind the target sequence and acts as one half of a stem structure that interacts non-covalently through hydrogen bonding, salt bridges, van der Waal's forces, etc.
  • Preferred embodiments utilize substantially complementary nucleic acids as the side chains.
  • the side chains of the primers upon hybridization of the primers to the target sequence, the side chains of the primers are brought into spatial proximity, and, if the side chains comprise nucleic acids as well, can also form side chain hybridization complexes.
  • At least one of the side chains of the primers comprises an activatable cross-linking agent, generally covalently attached to the side chain, that upon activation, results in a chemical cross-link or chemical ligation.
  • the activatible group may comprise any moiety that will allow cross-linking of the side chains, and include groups activated chemically, photonically and thermally, with photoactivatable groups being preferred.
  • a single activatable group on one of the side chains is enough to result in cross-linking via interaction to a functional group on the other side chain; in alternate embodiments, activatable groups are required on each side chain.
  • the reaction is subjected to conditions to allow for the disassocation of the hybridization complex, thus freeing up the target to serve as a template for the next ligation or cross-linking. In this way, signal amplification occurs, and can be detected as outlined herein.
  • the signal amplification technique is invasive cleavage technology, which is described in a number of patents and patent applications, including U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669, all of which are hereby incorporated by reference in their entirety.
  • a target nucleic acid is recognized by two distinct probes.
  • a first probe generally referred to herein as an “invader” probe, is substantially complementary to a first portion of the target nucleic acid.
  • a second probe generally referred to herein as a “signal probe”, is partially complementary to the target nucleic acid; the 3′ end of the signal oligonucleotide is substantially complementary to the target sequence while the 5′ end is non-complementary and preferably forms a single-stranded “tail” or “arm”.
  • the non-complementary end of the second probe preferably comprises a “generic” or “unique” sequence, frequently referred to herein as a “detection sequence”, that is used to indicate the presence or absence of the target nucleic acid, as described below.
  • the detection sequence of the second probe preferably comprises at least one detectable label, although as outlined herein, since this detection sequence can function as a target sequence for a capture probe, sandwich configurations utilizing label probes as described herein may also be done.
  • Hybridization of the first and second oligonucleotides near or adjacent to one another on the target nucleic acid forms a number of structures.
  • a forked cleavage structure forms and is a substrate of a nuclease which cleaves the detection sequence from the signal oligonucleotide.
  • the site of cleavage is controlled by the distance or overlap between the 3′ end of the invader oligonucleotide and the downstream fork of the signal oligonucleotide. Therefore, neither oligonucleotide is subject to cleavage when misaligned or when unattached to target nucleic acid.
  • the nuclease that recognizes the forked cleavage structure and catalyzes release of the tail is thermostable, thereby, allowing thermal cycling of the cleavage reaction, if desired.
  • Preferred nucleases derived from thermostable DNA polymerases that have been modified to have reduced synthetic activity which is an undesirable side-reaction during cleavage are disclosed in U.S. Pat. Nos. 5,719,028 and 5,843,669, hereby expressly by reference.
  • the synthetic activity of the DNA polymerase is reduced to a level where it does not interfere with detection of the cleavage reaction and detection of the freed tail.
  • the DNA polymerase has no detectable polymerase activity.
  • Examples of nucleases are those derived from Thermus aquaticus, Thermus flavus, or Thermus thermophilus.
  • thermostable structure-specific nucleases are Flap endonucleases (FENs) selected from FEN-1 or FEN-2 like (e.g. XPG and RAD2 nucleases) from Archaebacterial species, for example, FEN-1 from Methanococcus jannaschii, Pyrococcus furiosis, Pyrococcus woesei, and Archaeoglobus fulgidus. (U.S. Pat. No. 5,843,669 and Lyamichev et al. 1999. Nature Biotechnology 17:292-297; both of which are hereby expressly by reference).
  • FENs Flap endonucleases
  • the nuclease is AfuFEN1 or PfuFEN1 nuclease.
  • these nucleases require at least one overlapping nucleotide between the signal and invasive probes to recognize and cleave the 5′ end of the signal probe.
  • the 3′-terminal nucleotide of the invader oligonucleotide is not required to be complementary to the target nucleic acid.
  • mismatch of the signal probe one base upstream of the cleavage site prevents creation of the overlap and cleavage.
  • the specificity of the nuclease reaction allows single nucleotide polymorphism (SNP) detection from, for example, genomic DNA, as outlined below (Lyamichev et al.).
  • invasive cleavage technology is used.
  • Invasive cleavage technology is based on structure-specific nucleases that cleave nucleic acids in a site-specific manner. Two probes are used: an “invader” probe and a “signalling” probe, that adjacently hybridize to a target sequence with overlap.
  • the invader technology relies on complementarity at the overlap position where cleavage occurs.
  • the enzyme cleaves at the overlap, and releases the “tail” which may or may not be labeled. This can then be detected.
  • the InvaderTM technology is described in U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669, all of which are hereby incorporated by reference.
  • the invasive cleavage assay is preferably performed on an array format.
  • the signal probe has a detectable label, attached 5′ from the site of nuclease cleavage (e.g. within the detection sequence) and a capture tag, as described below (e.g. biotin or other hapten) 3′ from the site of nuclease cleavage.
  • the 3′ portion of the cleaved signal probe e.g. the the detection sequence
  • capture tag herein is a meant one of a pair of binding partners as described above, such as antigen/antibody pairs, digoxygenenin, dinitrophenol, etc.
  • the cleaved 5′ region e.g. the detection sequence, of the signal probe, comprises a label and is detected and optionally quantitated.
  • the cleaved 5′ region is hybridized to a probe on an array (capture probe) and optically detected. As described below, many signal probes can be analyzed in parallel by hybridization to their complementary probes in an array.
  • the invasive cleavage reaction is configured to utilize a fluorophore-quencher reaction.
  • a signalling probe comprising both a fluorophore and a quencher is used, with the fluorophore and the quencher on opposite sides of the cleavage site. As will be appreciated by those in the art, these will be positioned closely together. Thus, in the absence of cleavage, very little signal is seen due to the quenching reaction. After cleavage, however, the distance between the two is large, and thus fluorescence can be detected.
  • an assay complex comprising the target sequence, an invader probe, and a signalling probe, and the introduction of the cleavage enzyme, the cleavage of the complex results in the disassociation of the quencher from the complex, resulting in an increase in fluorescence.
  • suitable fluorophore-quencher pairs are as known in the art.
  • suitable quencher molecules comprise Dabcyl.
  • this system can be configured in a variety of conformations, as discussed in FIG. 4.
  • an additional enzymatic recognition step is introduced in the array capture procedure.
  • the cleaved signal probe binds to a capture probe to produce a double-stranded nucleic acid in the array.
  • the 3′ end of the cleaved signal probe is adjacent to the 5′ end of one strand of the capture probe, thereby, forming a substrate for DNA ligase (Broude et al. 1991. PNAS 91: 3072-3076).
  • Only correctly cleaved product is ligated to the capture probe.
  • Other incorrectly hybridized and non-cleaved signal probes are removed, for example, by heat denaturation, high stringency washes, and other methods that disrupt base pairing.
  • the signal amplification technique is CPT.
  • CPT technology is described in a number of patents and patent applications, including U.S. Pat. Nos. 5,011,769, 5,403,711, 5,660,988, and 4,876,187, and PCT published applications WO 95/05480, WO 95/1416, and WO 95/00667, and U.S. Ser. No. 09/014,304, all of which are expressly incorporated by reference in their entirety.
  • CPT may be described as follows.
  • a CPT primer (also sometimes referred to herein as a “scissile primer”), comprises two probe sequences separated by a scissile linkage.
  • the CPT primer is substantially complementary to the target sequence and thus will hybridize to it to form a hybridization complex.
  • the scissile linkage is cleaved, without cleaving the target sequence, resulting in the two probe sequences being separated.
  • the two probe sequences can thus be more easily disassociated from the target, and the reaction can be repeated any number of times.
  • the cleaved primer is then detected as outlined herein.
  • scissile linkage herein is meant a linkage within the scissile probe that can be cleaved when the probe is part of a hybridization complex, that is, when a double-stranded complex is formed. It is important that the scissile linkage cleave only the scissile probe and not the sequence to which it is hybridized (i.e. either the target sequence or a probe sequence), such that the target sequence may be reused in the reaction for amplification of the signal.
  • the scissile linkage is any connecting chemical structure which joins two probe sequences and which is capable of being selectively cleaved without cleavage of either the probe sequences or the sequence to which the scissile probe is hybridized.
  • the scissile linkage may be a single bond, or a multiple unit sequence. As will be appreciated by those in the art, a number of possible scissile linkages may be used.
  • the scissile linkage comprises RNA.
  • RNA This system, previously described in as outlined above, is based on the fact that certain double-stranded nucleases, particularly ribonucleases, will nick or excise RNA nucleosides from a RNA:DNA hybridization complex. Of particular use in this embodiment is RNAseH, Exo III, and reverse transcriptase.
  • the entire scissile probe is made of RNA, the nicking is facilitated especially when carried out with a double-stranded ribonuclease, such as RNAseH or Exo III.
  • RNA probes made entirely of RNA sequences are particularly useful because first, they can be more easily produced enzymatically, and second, they have more cleavage sites which are accessible to nicking or cleaving by a nicking agent, such as the ribonucleases.
  • scissile probes made entirely of RNA do not rely on a scissile linkage since the scissile linkage is inherent in the probe.
  • the methods of the invention may be used to detect mismatches, as is generally described in U.S. Pat. Nos. 5,660,988, and WO 95/14106, hereby expressly incorporated by reference. These mismatch detection methods are based on the fact that RNAseH may not bind to and/or cleave an RNA:DNA duplex if there are mismatches present in the sequence.
  • NA 1 and NA 2 are non-RNA nucleic acids, preferably DNA.
  • the mismatch is within the RNA:DNA duplex, but in some embodiments the mismatch is present in an adjacent sequence very close to the desired sequence, close enough to affect the RNAseH (generally within one or two bases).
  • the nucleic acid scissile linkage is designed such that the sequence of the scissile linkage reflects the particular sequence to be detected, i.e. the area of the putative mismatch.
  • the rate of generation of the released fragments is such that the methods provide, essentially, a yes/no result, whereby the detection of virtually any released fragment indicates the presence of the desired target sequence.
  • a minimal mismatch for example, a 1-, 2- or 3-base mismatch, or a 3-base deletion
  • the rate of generation of cleaved fragments, and/or the final amount of cleaved fragments is quantified to indicate the presence or absence of the target.
  • secondary and tertiary scissile probes may be particularly useful in this embodiment, as this can amplify the differences between a perfect match and a mismatch. These methods may be particularly useful in the determination of homozygotic or heterozygotic states of a patient.
  • the scissile linkage it is an important feature of the scissile linkage that its length is determined by the suspected difference between the target and the probe.
  • the scissile linkage is between 3 to 5 nucleotides in length, such that a suspected nucleotide difference from 1 nucleotide to 3 nucleotides is encompassed by the scissile linkage, and 0, 1 or 2 nucleotides are on either side of the difference.
  • the scissile linkage is nucleic acid
  • preferred embodiments utilize from 1 to about 100 nucleotides, with from about 2 to about 20 being preferred and from about 5 to about 10 being particularly preferred.
  • CPT may be done enzymatically or chemically. That is, in addition to RNAseH, there are several other cleaving agents which may be useful in cleaving RNA (or other nucleic acid) scissile bonds. For example, several chemical nucleases have been reported; see for example Sigman et al., Annu. Rev. Biochem. 1990, 59, 207-236; Sigman et al., Chem. Rev. 1993, 93, 2295-2316; Bashkin et al., J. Org. Chem. 1990, 55, 5125-5132; and Sigman et al., Nucleic Acids and Molecular Biology, vol. 3, F. Eckstein and D. M. J. Lilley (Eds), Springer-Verlag, Heidelberg 1989, pp. 13-27; all of which are hereby expressly incorporated by reference.
  • RNA hydrolysis is also an active area; see for example Chin, Acc. Chem. Res. 1991, 24, 145-152; Breslow et al., Tetrahedron, 1991, 47, 2365-2376; Anslyn et al., Angew. Chem. Int. Ed. Engl., 1997, 36, 432-450; and references therein, all of which are expressly incorporated by reference.
  • Reactive phosphate centers are also of interest in developing scissile linkages, see Hendry et al., Prog. Inorg. Chem.: Bioinorganic Chem. 1990, 31, 201-258 also expressly incorporated by reference.
  • RNA hydrolysis Current approaches to site-directed RNA hydrolysis include the conjugation of a reactive moiety capable of cleaving phosphodiester bonds to a recognition element capable of sequence-specifically hybridizing to RNA. In most cases, a metal complex is covalently attached to a DNA strand which forms a stable heteroduplex. Upon hybridization, a Lewis acid is placed in close proximity to the RNA backbone to effect hydrolysis; see Magda et al., J. Am. Chem. Soc. 1994, 116, 7439; Hall et al., Chem. Biology 1994, 1, 185-190; Bashkin et al., J. Am. Chem. Soc.
  • the scissile linkage is not necessarily RNA.
  • chemical cleavage moieties may be used to cleave basic sites in nucleic acids; see Belmont, et al.,New J. Chem. 1997, 21, 47-54; and references therein, all of which are expressly incorporated herein by reference.
  • photocleavable moieties for example, using transition metals, may be used; see Moucheron, et al., Inorg. Chem. 1997, 36, 584-592, hereby expressly by reference.
  • the first step of the CPT method requires hybridizing a primary scissile primer (also called a primary scissile probe) to the target. This is preferably done at a temperature that allows both the binding of the longer primary probe and disassociation of the shorter cleaved portions of the primary probe, as will be appreciated by those in the art. As outlined herein, this may be done in solution, or either the target or one or more of the scissile probes may be attached to a solid support. For example, it is possible to utilize “anchor probes” on a solid support which are substantially complementary to a portion of the target sequence, preferably a sequence that is not the same sequence to which a scissile probe will bind.
  • a preferred embodiment has one or more of the scissile probes attached to a solid support such as a bead.
  • the soluble target diffuses to allow the formation of the hybridization complex between the soluble target sequence and the support-bound scissile probe.
  • preferred methods utilize cutting or shearing techniques to cut the nucleic acid sample containing the target sequence into a size that will allow sufficient diffusion of the target sequence to the surface of a bead. This may be accomplished by shearing the nucleic acid through mechanical forces (e.g. sonication) or by cleaving the nucleic acid using restriction endonucleases. Alternatively, a fragment containing the target may be generated using polymerase, primers and the sample as a template, as in polymerase chain reaction (PCR). In addition, amplification of the target using PCR or LCR or related methods may also be done; this may be particularly useful when the target sequence is present in the sample at extremely low copy numbers. Similarly, numerous techniques are known in the art to increase the rate of mixing and hybridization including agitation, heating, techniques that increase the overall concentration such as precipitation, drying, dialysis, centrifugation, electrophoresis, magnetic bead concentration, etc.
  • the scissile probes are introduced in a molar excess to their targets (including both the target sequence or other scissile probes, for example when secondary or tertiary scissile probes are used), with ratios of scissile probe:target of at least about 100:1 being preferred, at least about 1000:1 being particularly preferred, and at least about 10,000:1 being especially preferred. In some embodiments the excess of probe:target will be much greater. In addition, ratios such as these may be used for all the amplification techniques outlined herein.
  • the complex is subjected to cleavage conditions. As will be appreciated, this depends on the composition of the scissile probe; if it is RNA, RNAseH is introduced. It should be noted that under certain circumstances, such as is generally outlined in WO 95/00666 and WO 95/00667, hereby incorporated by reference, the use of a double-stranded binding agent such as RNAseH may allow the reaction to proceed even at temperatures above the Tm of the primary probe:target hybridization complex. Accordingly, the addition of scissile probe to the target can be done either first, and then the cleavage agent or cleavage conditions introduced, or the probes may be added in the presence of the cleavage agent or conditions.
  • the cleavage conditions result in the separation of the two (or more) probe sequences of the primary scissile probe. As a result, the shorter probe sequences will no longer remain hybridized to the target sequence, and thus the hybridization complex will disassociate, leaving the target sequence intact.
  • the optimal temperature for carrying out the CPT reactions is generally from about 5° C. to about 25° C. below the melting temperatures of the probe:target hybridization complex. This provides for a rapid rate of hybridization and high degree of specificity for the target sequence.
  • the Tm of any particular hybridization complex depends on salt concentration, G-C content, and length of the complex, as is known in the art and described herein.
  • nucleases are generally removed from the sample during the isolation of the DNA by heating or extraction procedures.
  • a number of inhibitors of single-stranded nucleases such as vanadate, inhibitors it-ACE and RNAsin, a placental protein, do not affect the activity of RNAseH. This may not be necessary depending on the purity of the RNAseH and/or the target sample.
  • each molecule of the target sequence will turnover between 100 and 1000 times in this period, depending on the length and sequence of the probe, the specific reaction conditions, and the cleavage method. For example, for each copy of the target sequence present in the test sample 100 to 1000 molecules will be cleaved by RNAseH. Higher levels of amplification can be obtained by allowing the reaction to proceed longer, or using secondary, tertiary, or quaternary probes, as is outlined herein.
  • the uncleaved scissile probes Upon completion of the reaction, generally determined by time or amount of cleavage, the uncleaved scissile probes must be removed or neutralized prior to detection, such that the uncleaved probe does not bind to a detection probe, causing false positive signals. This may be done in a variety of ways, as is generally described below.
  • the separation is facilitated by the use of beads containing the primary probe.
  • beads containing the primary probe When the scissile probes are attached to beads, removal of the beads by filtration, centrifugation, the application of a magnetic field, electrostatic interactions for charged beads, adhesion, etc., results in the removal of the uncleaved probes.
  • the separation is based on strong acid precipitation. This is useful to separate long (generally greater than 50 nucleotides) from smaller fragments (generally about 10 nucleotides).
  • a strong acid such as trichloroacetic acid
  • the solution can be centrifuged or filtered to remove the precipitate, and the cleaved probe sequences can be quantitated.
  • the scissile probe contains both a detectable label and an affinity binding ligand or moiety, such that an affinity support is used to carry out the separation.
  • the detectable label used for detection is not on the same probe sequence that contains the affinity moiety, such that removal of the uncleaved probe, and the cleaved probe containing the affinity moiety, does not remove all the detectable labels.
  • the scissile probe may contain a capture tag; the binding partner of the capture tag is attached to a solid support such as glass beads, latex beads, dextrans, etc. and used to pull out the uncleaved probes, as is known in the art.
  • the cleaved probe sequences, which do not contain the capture tag remain in solution and then can be detected as outlined below.
  • a separation sequence of nucleic acid is included in the scissile probe, which is not cleaved during the reaction.
  • a nucleic acid complementary to the separation sequence is attached to a solid support such as a bead and serves as a catcher sequence.
  • the separation sequence is added to the scissile probes, and is not recognized by the target sequence, such that a generalized catcher sequence may be utilized in a variety of assays.
  • the cleaved probe is bound to a capture probe, either directly or indirectly, and the label is detected.
  • no higher order probes are used, and detection is based on the probe sequence(s) of the primary primer.
  • at least one, and preferably more, secondary probes are used; the secondary probes hybridize to the domains of the cleavage probes; etc.
  • CPT requires, again in no particular order, a first CPT primer comprising a first probe sequence, a scissile linkage and a second probe sequence; and a cleavage agent.
  • CPT results in the generation of a large amount of cleaved primers, which then can be detected as outlined below.
  • direct detection as used in this context, as for the other amplification strategies outlined herein, requires the incorporation of a label, in this case a detectable label, preferably an optical label such as a fluorophore, into the target sequence, with detection proceeding as outlined below.
  • a label in this case a detectable label, preferably an optical label such as a fluorophore
  • the label(s) may be incorporated in three ways: (1) the primers comprise the label(s), for example attached to the base, a ribose, a phosphate, or to analogous structures in a nucleic acid analog; (2) modified nucleosides are used that are modified at either the base or the ribose (or to analogous structures in a nucleic acid analog) with the label(s); these label-modified nucleosides are then converted to the triphosphate form and are incorporated into the newly synthesized strand by a polymerase; (3) modified nucleotides are used that comprise a functional group that can be used to add a detectable label; or (4) modified primers are used that comprise a functional group that can be used to add a detectable label. Any of these methods result in a newly synthesized strand that comprises labels, that can be directly detected as outlined below.
  • the modified strands comprise a detection label.
  • detection label or “detectable label” herein is meant a moiety that allows detection. This may be a primary label or a secondary label.
  • the detection label is a primary label.
  • a primary label is one that can be directly detected, such as a fluorophore.
  • labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic, electrical, thermal labels; and c) colored or luminescent dyes. Labels can also include enzymes (horseradish peroxidase, etc.) and magnetic particles. Preferred labels include chromophores or phosphors but are preferably fluorescent dyes.
  • Suitable dyes for use in the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, alexa dyes, phycoerythin, bodipy, and others described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference.
  • fluorescent lanthanide complexes including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, alex
  • a secondary detectable label is used.
  • detection labels may be primary labels (i.e. directly detectable) or secondary labels (indirectly detectable).
  • a secondary label is one that is indirectly detected; for example, a secondary label can bind or react with a primary label for detection, or may allow the separation of the compound comprising the secondary label from unlabeled materials, etc.
  • Secondary labels find particular use in systems requiring separation of labeled and unlabeled probes, such as SBE reactions. Secondary labels include, but are not limited to, one of a binding partner pair; chemically modifiable moieties; nuclease inhibitors, etc.
  • the secondary label is a binding partner pair.
  • the label may be a hapten or antigen, which will bind its binding partner.
  • the binding partner can be attached to a solid support to allow separation of extended and non-extended primers.
  • suitable binding partner pairs include, but are not limited to: antigens (such as proteins (including peptides)) and antibodies (including fragments thereof (FAbs, etc.)); proteins and small molecules, including biotin/streptavidin; enzymes and substrates or inhibitors; other protein-protein interacting pairs; receptor-ligands; and carbohydrates and their binding partners. Nucleic acid-nucleic acid binding proteins pairs are also useful. In general, the smaller of the pair is attached to the NTP for incorporation into the extension primer.
  • the binding partner pair comprises biotin or imino-biotin and streptavidin.
  • Imino-biotin is particularly preferred as imino-biotin disassociates from streptavidin in pH 4.0 buffer while biotin requires harsh denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or 90% formamide at 95° C.).
  • the binding partner pair comprises a primary detection label (for example, attached to the NTP and therefore to the extended primer) and an antibody that will specifically bind to the primary detection label.
  • a primary detection label for example, attached to the NTP and therefore to the extended primer
  • an antibody that will specifically bind to the primary detection label.
  • specifically bind herein is meant that the partners bind with specificity sufficient to differentiate between the pair and other components or contaminants of the system. The binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding.
  • the dissociation constants of the pair will be less than about 10 ⁇ 4 -10 ⁇ 6 M ⁇ 1 , with less than about 10 ⁇ 5 to 10 ⁇ 9 M ⁇ 1 being preferred and less than about 10 ⁇ 7 -10 ⁇ 9 M ⁇ 1 being particularly preferred.
  • the other half of the binding pair is attached to a solid support.
  • the solid support may be any as described herein for substrates and microspheres, and the form is preferably microspheres as well; for example, a preferred embodiment utilizes magnetic beads that can be easily introduced to the sample and easily removed, although any affinity chromatography formats may be used as well. Standard methods are used to attach the binding partner to the solid support, and can include direct or indirect attachment methods. For example, biotin labeled antibodies to fluorophores can be attached to streptavidin coated magnetic beads.
  • the extended primers comprise a binding partner that is contacted with its binding partner under conditions wherein the extended primers are separated from the unextended primers. These extended primers can then be added to the array comprising capture probes as described herein.
  • the secondary label is a chemically modifiable moiety.
  • labels comprising reactive functional groups are incorporated into the nucleic acid.
  • the functional group can then be subsequently labeled with a primary label.
  • Suitable functional groups include, but are not limited to, amino groups, carboxy groups, maleimide groups, oxo groups and thiol groups, with amino groups and thiol groups being particularly preferred.
  • the unextended or unreacted primers from the assay mixture, and particularly from the array, as unextended primers will compete with the extended (labeled) primers in binding to capture probes, thereby diminishing the signal.
  • concentration of the unextended primers relative to the extended primer may be relatively high, since a large excess of primer is usually required to generate efficient primer annealing. Accordingly, a number of different techniques may be used to facilitate the removal of unextended primers. While the discussion below applies specifically to SBE, these techniques may be used in any of the methods described herein.
  • the NTPs (or, in the case of other methods, one or more of the probes) comprise a secondary detectable label that can be used to separate extended and non-extended primers.
  • detection labels may be primary labels (i.e. directly detectable) or secondary labels (indirectly detectable).
  • a secondary label is one that is indirectly detected; for example, a secondary label can bind or react with a primary label for detection, or may allow the separation of the compound comprising the secondary label from unlabeled materials, etc.
  • Secondary labels find particular use in systems requiring separation of labeled and unlabeled probes, such as SBE, OLA, invasive cleavage, etc. reactions; in addition, these techniques may be used with many of the other techniques described herein.
  • Secondary labels include, but are not limited to, one of a binding partner pair; chemically modifiable moieties; nuclease inhibitors, etc.
  • the secondary label is a binding partner pair.
  • the label may be a hapten or antigen, Which will bind its binding partner (generally attached to a solid support) and thus allow separation of extended and non-extended primers.
  • suitable binding partner pairs include, but are not limited to: antigens (such as proteins (including peptides)) and antibodies (including fragments thereof (FAbs, etc.)); proteins and small molecules, including biotin/streptavidin and digoxygenin and antibodies; enzymes and substrates or inhibitors; other protein-protein interacting pairs; receptor-ligands; and carbohydrates and their binding partners, are also suitable binding pairs. Nucleic acid-nucleic acid binding proteins pairs are also useful. In general, the smaller of the pair is attached to the NTP (or the probe) for incorporation into the extension primer.
  • the binding partner pair comprises biotin or imino-biotin and streptavidin.
  • Imino-biotin is particularly preferred when the methods require the later separation of the pair, as imino-biotin disassociates from streptavidin in pH 4.0 buffer while biotin requires harsh denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or 90% formamide at 95° C.).
  • streptavidin/biotin systems can be used to separate unreacted and reacted probes (for example in SBE, invasive cleavage, etc.).
  • the addition of streptavidin to a nucleic acid greatly increases its size, as well as changes its physical properties, to allow more efficient separation techniques.
  • the mixtures can be size fractionated by exclusion chromatography, affinity chromatography, filtration or differential precipitation.
  • an 3′ exonuclease may be added to a mixture of 3′ labeled biotin/streptavidin; only the unreacted oligonucleotides will be degraded.
  • the exonuclease and the streptavidin can be degraded using a protease such as proteinase K.
  • the surviving nucleic acids i.e. those that were biotinylated are then hybridized to the array.
  • the binding partner pair comprises a primary detection label (attached to the NTP and therefore to the extended primer) and an antibody that will specifically bind to the primary detection label.
  • specifically bind herein is meant that the partners bind with specificity sufficient to differentiate between the pair and other components or contaminants of the system. The binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding.
  • the dissociation constants of the pair will be less than about 10 ⁇ 4 -10 ⁇ 6 M ⁇ 1 , with less than about 10 ⁇ 5 to 10 ⁇ 9 M ⁇ 1 being preferred and less than about 10 ⁇ 7 -10 ⁇ 9 M ⁇ 1 being particularly preferred.
  • the other half of the binding pair is attached to a solid support.
  • the solid support may be any as described herein for substrates and microspheres, and the form is preferably microspheres as well; for example, a preferred embodiment utilizes magnetic beads that can be easily introduced to the sample and easily removed, although any affinity chromatography formats may be used as well. Standard methods are used to attach the binding partner to the solid support, and can include direct or indirect attachment methods. For example, biotin labeled antibodies to fluorophores can be attached to streptavidin coated magnetic beads.
  • the extended primers comprise a binding member that is contacted with its binding partner under conditions wherein the extended primers are separated from the unextended primers. These extended primers can then be added to the array comprising capture probes as described herein.
  • the secondary label is a chemically modifiable moiety.
  • labels comprising reactive functional groups are incorporated into the nucleic acid.
  • the secondary label is a nuclease inhibitor.
  • the chain-terminating NTPs are chosen to render extended primers resistant to nucleases, such as 3′-exonucleases. Addition of an exonuclease will digest the non-extended primers leaving only the extended primers to bind to the capture probes on the array. This may also be done with OLA, wherein the ligated probe will be protected but the unprotected ligation probe will be digested.
  • suitable 3′-exonucleases include, but are not limited to, exo I, exo III, exo VII, etc.
  • the signal amplification technique is a “sandwich” assay, as is generally described in U.S. Ser. No. 60/073,011 and in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporated by reference.
  • sandwich assays do not result in the alteration of primers, sandwich assays can be considered signal amplification techniques since multiple signals (i.e. label probes) are bound to a single target, resulting in the amplification of the signal.
  • Sandwich assays may be used when the target sequence does not contain a label; or when adapters are used, as outlined below.
  • the sandwich assays can be used for the detection of primary target sequences (e.g. from a patient sample), or as a method to detect the product of an amplification reaction as outlined above; thus for example, any of the newly synthesized strands outlined above, for example using PCR, LCR, NASBA, SDA, etc., may be used as the “target sequence” in a sandwich assay.
  • the systems of the invention may take on a large number of different configurations.
  • capture probes and optionally either capture extender probes (sometimes referred to as “adapter sequences” herein).
  • capture extender probes may be used, that allow a “universal” surface, i.e. a surface containing a single type of capture probe that can be used to detect any target sequence.
  • Capture extender probes have a first portion that will hybridize to all or part of the capture probe, and a second portion that will hybridize to a first portion of the target sequence. This then allows the generation of customized soluble probes, which as will be appreciated by those in the art is generally simpler and less costly. As shown herein, two capture extender probes may be used. This has generally been done to stabilize assay complexes for example when the target sequence is large, or when large amplifier probes (particularly branched or dendrimer amplifier probes) are used.
  • Detection of the amplification reactions of the invention is preferably done by detecting assay complexes comprising detectable labels, which can be attached to the assay complex in a variety of ways, as is more fully described below.
  • an amplifier probe is hybridized to the target sequence, either directly, or through the use of one or more label extender probes, which serves to allow “generic” amplifier probes to be made. As for all the steps outlined herein, this may be done simultaneously with capturing, or sequentially.
  • the amplifier probe contains a multiplicity of amplification sequences, although in some embodiments, as described below, the amplifier probe may contain only a single amplification sequence, or at least two amplification sequences.
  • the amplifier probe may take on a number of different forms; either a branched conformation, a dendrimer conformation, or a linear “string” of amplification sequences.
  • Label probes comprising detectable labels (preferably but not required to be fluorophores) then hybridize to the amplification sequences (or in some cases the label probes hybridize directly to the target sequence), and the labels detected, as is more fully outlined below.
  • compositions comprising an amplifier probe.
  • amplifier probe or “nucleic acid multimer” or “amplification multimer” or grammatical equivalents herein is meant a nucleic acid probe that is used to facilitate signal amplification.
  • Amplifier probes comprise at least a first single-stranded nucleic acid probe sequence, as defined below, and at least one single-stranded nucleic acid amplification sequence, with a multiplicity of amplification sequences being preferred.
  • Amplifier probes comprise a first probe sequence that is used, either directly or indirectly, to hybridize to the target sequence. That is, the amplifier probe itself may have a first probe sequence that is substantially complementary to the target sequence, or it has a first probe sequence that is substantially complementary to a portion of an additional probe, in this case called a label extender probe, that has a first portion that is substantially complementary to the target sequence. In a preferred embodiment, the first probe sequence of the amplifier probe is substantially complementary to the target sequence.
  • the first probe sequence is of a length sufficient to give specificity and stability.
  • the probe sequences of the invention that are designed to hybridize to another nucleic acid are at least about 5 nucleosides long, with at least about 10 being preferred and at least about 15 being especially preferred.
  • the amplifier probe provides an amplification of signal due to a multiplicity of labelling events, and several different amplifier probes, each with this multiplicity of labels, for each target sequence is used.
  • preferred embodiments utilize at least two different pools of amplifier probes, each pool having a different probe sequence for hybridization to different portions of the target sequence; the only real limitation on the number of different amplifier probes will be the length of the original target sequence.
  • the different amplifier probes contain different amplification sequences, although this is generally not preferred.
  • the amplifier probe does not hybridize to the sample target sequence directly, but instead hybridizes to a first portion of a label extender probe.
  • a label extender probe This is particularly useful to allow the use of “generic” amplifier probes, that is, amplifier probes that can be used with a variety of different targets. This may be desirable since several of the amplifier probes require special synthesis techniques. Thus, the addition of a relatively short probe as a label extender probe is preferred.
  • the first probe sequence of the amplifier probe is substantially complementary to a first portion or domain of a first label extender single-stranded nucleic acid probe.
  • the label extender probe also contains a second portion or domain that is substantially complementary to a portion of the target sequence.
  • Both of these portions are preferably at least about 10 to about 50 nucleotides in length, with a range of about 15 to about 30 being preferred.
  • the terms “first” and “second” are not meant to confer an orientation of the sequences with respect to the 5′-3′ orientation of the target or probe sequences.
  • the first portion may be located either 5′ to the second portion, or 3′ to the second portion.
  • the order of probe sequences are generally shown from left to right.
  • more than one label extender probe-amplifier probe pair may be used, that is, n is more than 1. That is, a plurality of label extender probes may be used, each with a portion that is substantially complementary to a different portion of the target sequence; this can serve as another level of amplification. Thus, a preferred embodiment utilizes pools of at least two label extender probes, with the upper limit being set by the length of the target sequence.
  • more than one label extender probe is used with a single amplifier probe to reduce non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697, incorporated by reference herein.
  • a first portion of the first label extender probe hybridizes to a first portion of the target sequence
  • the second portion of the first label extender probe hybridizes to a first probe sequence of the amplifier probe.
  • a first portion of the second label extender probe hybridizes to a second portion of the target sequence
  • the second portion of the second label extender probe hybridizes to a second probe sequence of the amplifier probe.
  • the label extender probes may interact with a preamplifier probe, described below, rather than the amplifier probe directly.
  • a preferred embodiment utilizes several different amplifier probes, each with first probe sequences that will hybridize to a different portion of the label extender probe.
  • the different amplifier probes contain different amplification sequences, although this is generally not preferred.
  • the amplifier probe also comprises at least one amplification sequence.
  • An “amplification sequence” or “amplification segment” or grammatical equivalents herein is meant a sequence that is used, either directly or indirectly, to bind to a first portion of a label probe as is more fully described below.
  • the amplifier probe comprises a multiplicity of amplification sequences, with from about 3 to about 1000 being preferred, from about 10 to about 100 being particularly preferred, and about 50 being especially preferred. In some cases, for example when linear amplifier probes are used, from 1 to about 20 is preferred with from about 5 to about 10 being particularly preferred.
  • the amplification sequences may be linked to each other in a variety of ways, as will be appreciated by those in the art. They may be covalently linked directly to each other, or to intervening sequences or chemical moieties, through nucleic acid linkages such as phosphodiester bonds, PNA bonds, etc., or through interposed linking agents such amino acid, carbohydrate or polyol bridges, or through other cross-linking agents or binding partners.
  • the site(s) of linkage may be at the ends of a segment, and/or at one or more internal nucleotides in the strand.
  • the amplification sequences are attached via nucleic acid linkages.
  • branched amplifier probes are used, as are generally described in U.S. Pat. No. 5,124,246, hereby incorporated by reference. Branched amplifier probes may take on “fork-like” or “comb-like” conformations. “Fork-like” branched amplifier probes generally have three or more oligonucleotide segments emanating from a point of origin to form a branched structure. The point of origin may be another nucleotide segment or a multifunctional molecule to which at least three segments can be covalently or tightly bound. “Comb-like” branched amplifier probes have a linear backbone with a multiplicity of sidechain oligonucleotides extending from the backbone.
  • the pendant segments will normally depend from a modified nucleotide or other organic moiety having the appropriate functional groups for attachment of oligonucleotides.
  • a large number of amplification sequences are available for binding, either directly or indirectly, to detection probes.
  • these structures are made as is known in the art, using modified multifunctional nucleotides, as is described in U.S. Pat. Nos. 5,635,352 and 5,124,246, among others.
  • dendrimer amplifier probes are used, as are generally described in U.S. Pat. No. 5,175,270, hereby expressly incorporated by reference.
  • Dendrimeric amplifier probes have amplification sequences that are attached via hybridization, and thus have portions of double-stranded nucleic acid as a component of their structure.
  • the outer surface of the dendrimer amplifier probe has a multiplicity of amplification sequences.
  • linear amplifier probes are used, that have individual amplification sequences linked end-to-end either directly or with short intervening sequences to form a polymer. As with the other amplifier configurations, there may be additional sequences or moieties between the amplification sequences. In one embodiment, the linear amplifier probe has a single amplification sequence.
  • the amplifier probe may be totally linear, totally branched, totally dendrimeric, or any combination thereof.
  • the amplification sequences of the amplifier probe are used, either directly or indirectly, to bind to a label probe to allow detection.
  • the amplification sequences of the amplifier probe are substantially complementary to a first portion of a label probe.
  • amplifier extender probes are used, that have a first portion that binds to the amplification sequence and a second portion that binds to the first portion of the label probe.
  • compositions of the invention may include “preamplifier” molecules, which serves a bridging moiety between the label extender molecules and the amplifier probes. In this way, more amplifier and thus more labels are ultimately bound to the detection probes.
  • Preamplifier molecules may be either linear or branched, and typically contain in the range of about 30-3000 nucleotides.
  • label probes are either substantially complementary to an amplification sequence or to a portion of the target sequence.
  • Detection of the amplification reactions of the invention is done by detecting assay complexes comprising labels.
  • Detection of the amplified products described above preferably employs arrays, as defined herein.
  • the arrays are preferably high density arrays that can allow simultaneous analysis, i.e. parallel rather than serial processing, on a number of samples. This is preferably done by forming an “array of arrays”, i.e. a composite array comprising a plurality of individual arrays, that is configured to allow processing of multiple samples, as is generally outlined in U.S. Ser. No. 09/256,943, hereby expressly incorporated by reference.
  • each individual array is present within each well of a microtiter plate.
  • microtiter plate depending on the size of the microtiter plate and the size of the individual array, very high numbers of assays can be run simultaneously; for example, using individual arrays of 2,000 and a 96 well microtiter plate, 192,000 experiments can be done at once; the same arrays in a 384 microtiter plate yields 768,000 simultaneous experiments, and a 1536 microtiter plate gives 3,072,000 experiments.
  • the array of array compositions of the invention can be configured in several ways.
  • a “one component” system is used. That is, a first substrate comprising a plurality of assay locations (sometimes also referred to herein as “assay wells”), such as a microtiter plate, is configured such that each assay location contains an individual array. That is, the assay location and the array location are the same.
  • the plastic material of the microtiter plate can be formed to contain a plurality of “bead wells” in the bottom of each of the assay wells. Beads containing the capture probes of the invention can then be loaded into the bead wells in each assay location as is more fully described below.
  • a “two component” system can be used.
  • the individual arrays are formed on a second substrate, which then can be fitted or “dipped” into the first microtiter plate substrate.
  • a preferred embodiment utilizes fiber optic bundles as the individual arrays, generally with “bead wells” etched into one surface of each individual fiber, such that the beads containing the capture probes are loaded onto the end of the fiber optic bundle.
  • the composite array thus comprises a number of individual arrays that are configured to fit within the wells of a microtiter plate.
  • the present invention is generally based on previous work comprising a bead-based analytic chemistry system in which beads, also termed microspheres, carrying different chemical functionalities are distributed on a substrate comprising a patterned surface of discrete sites that can bind the individual microspheres.
  • the beads are generally put onto the substrate randomly, and thus several different methodologies can be used to “decode” the arrays.
  • unique optical signatures are incorporated into the beads, generally fluorescent dyes, that could be used to identify the chemical functionality on any particular bead. This allows the synthesis of the nucleic acids to be divorced from their placement on an array, i.e. the capture probes may be synthesized on the beads, and then the beads are randomly distributed on a patterned surface.
  • the beads are first coded with an optical signature, this means that the array can later be “decoded”, i.e. after the array is made, a correlation of the location of an individual site on the array with the probe at that particular site can be made. This means that the beads may be randomly distributed on the array, a fast and inexpensive process as compared to either the in situ synthesis or spotting techniques of the prior art. These methods are generally outlined in PCTs US98/05025 and US99/14387 and U.S. Ser. Nos. 08/818,199 and 09/151,877, all of which are expressly incorporated herein by reference.
  • the drawback to these methods is that for a very high density array, the system requires a large number of different optical signatures, which may be difficult or time-consuming to utilize. Accordingly, the present invention also provides several improvements over these methods, generally directed to methods of coding and decoding the arrays. That is, as will be appreciated by those in the art, the placement of the probes is generally random, and thus a coding/decoding system is required to identify the probes at each location in the array.
  • DBL decoding binding ligand
  • IBLs identifier binding ligands
  • positional decoding for example by either targeting the placement of beads (for example by using photoactivatible or photocleavable moieties to allow the selective addition of beads to particular locations), or by using either sub-bundles or selective loading of the sites, as are more fully outlined below; c) selective decoding, wherein only those beads that bind to a target are decoded; or d) combinations of any of these.
  • this decoding may occur for all the beads, or only for those that bind a particular target analyte. Similarly, this may occur either prior to or after addition of a target analyte.
  • the array is exposed to samples containing the target sequences, such as, the products of amplification reactions described above, although as outlined below, this can be done prior to or during the analysis as well.
  • the target sequences will bind to the capture probes as is more fully outlined below, and results (in the case of optical labels) in a change in the optical signal of a particular bead.
  • decoding can use optical signatures, decoding binding ligands that are added during a decoding step, or a combination of these methods.
  • the decoding binding ligands will bind either to a distinct identifier binding ligand partner that is placed on the beads, or to the capture probes, with the latter being preferred.
  • the decoding binding ligands are either directly or indirectly labeled, and thus decoding occurs by detecting the presence of the label.
  • the present invention provides composite array compositions comprising at least a first substrate with a surface comprising a plurality of assay locations.
  • array herein is meant a plurality of candidate agents in an array format; the size of the array will depend on the composition and end use of the array. Arrays containing from about 2 different probes (i.e. different beads) to many millions can be made, with very large fiber optic arrays being possible. Generally, the array will comprise from two to as many as a billion or more per square cm, depending on the size of the beads and the substrate, as well as the end use of the array, thus very high density, high density, moderate density, low density and very low density arrays may be made.
  • Preferred ranges for very high density arrays are from about 10,000,000 to about 2,000,000,000, (with all numbers being per square centimeter) with from about 100,000,000 to about 1,000,000,000 being preferred.
  • High density arrays range about 100,000 to about 10,000,000, with from about 1,000,000 to about 5,000,000 being particularly preferred.
  • Moderate density arrays range from about 10,000 to about 100,000 being particularly preferred, and from about 20,000 to about 50,000 being especially preferred.
  • Low density arrays are generally less than 10,000, with from about 1,000 to about 5,000 being preferred.
  • Very low density arrays are less than 1,000, with from about 10 to about 1000 being preferred, and from about 100 to about 500 being particularly preferred.
  • the compositions of the invention may not be in array format; that is, for some embodiments, compositions comprising a single bioactive agent may be made as well.
  • multiple substrates may be used, either of different or identical compositions. Thus for example, large arrays may comprise a plurality of smaller substrates.
  • one advantage of the present compositions is that particularly through the use of fiber optic technology, extremely high density arrays can be made.
  • beads of 200 ⁇ m or less can be used, and very small fibers are known, it is possible to have as many as 250,000 or more (in some instances, 1 million) different fibers and beads in a 1 mm 2 fiber optic bundle, with densities of greater than 15,000,000 individual beads and fibers (again, in some instances as many as 25-50 million) per 0.5 cm 2 obtainable.
  • composite array or “combination array” or grammatical equivalents herein is meant a plurality of individual arrays, as outlined above. Generally the number of individual arrays is set by the size of the microtiter plate used; thus, 96 well, 384 well and 1536 well microtiter plates utilize composite arrays comprising 96, 384 and 1536 individual arrays, although as will be appreciated by those in the art, not each microtiter well need contain an individual array. It should be noted that the composite arrays can comprise individual arrays that are identical, similar or different. That is, in some embodiments, it may be desirable to do the same 2,000 assays on 96 different samples; alternatively, doing 192,000 experiments on the same sample (i.e.
  • each row or column of the composite array could be the same, for redundancy/quality control.
  • the random nature of the arrays may mean that the same population of beads may be added to two different surfaces, resulting in substantially similar but perhaps not identical arrays.
  • substrate or “solid support” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of beads and is amenable to at least one detection method. As will be appreciated by those in the art, the number of possible substrates is very large.
  • Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers.
  • plastics including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.
  • polysaccharides such as polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.
  • polysaccharides such as polypropylene, polyethylene, polybutylene, polyurethanes
  • the substrate is flat (planar), although as will be appreciated by those in the art, other configurations of substrates may be used as well; for example, three dimensional configurations can be used, for example by embedding the beads in a porous block of plastic that allows sample access to the beads and using a confocal microscope for detection. Similarly, the beads may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
  • Preferred substrates include optical fiber bundles as discussed below, and flat planar substrates such as glass, polystyrene and other plastics and acrylics.
  • the first substrate comprises a surface comprising a plurality of assay locations, i.e. the location where the assay for the detection of a target analyte will occur.
  • the assay locations are generally physically separated from each other, for example as assay wells in a microtiter plate, although other configurations (hydrophobicity/hydrophilicity, etc.) can be used to separate the assay locations.
  • the second substrate is an optical fiber bundle or array, as is generally described in U.S. Ser. Nos. 08/944,850 and 08/519,062, PCT US98/05025, and PCT US98/09163, all of which are expressly incorporated herein by reference.
  • Preferred embodiments utilize preformed unitary fiber optic arrays.
  • preformed unitary fiber optic array herein is meant an array of discrete individual fiber optic strands that are co-axially disposed and joined along their lengths. The fiber strands are generally individually clad.
  • the fibers are not individually physically manipulatable; that is, one strand generally cannot be physically separated at any point along its length from another fiber strand.
  • the array comprises a plurality of discrete sites.
  • the assay location is the same as the array location, as described herein.
  • the array location is fitted into the assay location separately.
  • at least one surface of the substrate is modified to contain discrete, individual sites for later association of microspheres. These sites may comprise physically altered sites, i.e. physical configurations such as wells or small depressions in the substrate that can retain the beads, such that a microsphere can rest in the well, or the use of other forces (magnetic or compressive), or chemically altered or active sites, such as chemically functionalized sites, electrostatically altered sites, hydrophobically/hydrophilically functionalized sites, spots of adhesive, etc.
  • the sites may be a pattern, i.e. a regular design or configuration, or randomly distributed.
  • a preferred embodiment utilizes a regular pattern of sites such that the sites may be addressed in the X-Y coordinate plane.
  • “Pattern” in this sense includes a repeating unit cell, preferably one that allows a high density of beads on the substrate.
  • these sites may not be discrete sites. That is, it is possible to use a uniform surface of adhesive or chemical functionalities, for example, that allows the attachment of beads at any position. That is, the surface of the substrate is modified to allow attachment of the microspheres at individual sites, whether or not those sites are contiguous or non-contiguous with other sites.
  • the surface of the substrate may be modified such that discrete sites are formed that can only have a single associated bead, or alternatively, the surface of the substrate is modified and beads may go down anywhere, but they end up at discrete sites.
  • the surface of the substrate is modified to contain wells, i.e. depressions in the surface of the substrate.
  • wells i.e. depressions in the surface of the substrate.
  • This may be done as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques.
  • the technique used will depend on the composition and shape of the substrate.
  • a preferred method utilizes molding techniques that form the bead wells in the bottom of the assay wells in a microtiter plate.
  • physical alterations are made in a surface of the substrate to produce the sites.
  • the surface of the substrate is a terminal end of the fiber bundle, as is generally described in 08/818,199 and 09/151,877, both of which are hereby expressly incorporated by reference.
  • wells are made in a terminal or distal end of a fiber optic bundle comprising individual fibers.
  • the cores of the individual fibers are etched, with respect to the cladding, such that small wells or depressions are formed at one end of the fibers. The required depth of the wells will depend on the size of the beads to be added to the wells.
  • the microspheres are non-covalently associated in the wells, although the wells may additionally be chemically functionalized as is generally described below, cross-linking agents may be used, or a physical barrier may be used, i.e. a film or membrane over the beads.
  • the surface of the substrate is modified to contain chemically modified sites, that can be used to attach, either covalently or non-covalently, the microspheres of the invention to the discrete sites or locations on the substrate.
  • “Chemically modified sites” in this context includes, but is not limited to, the addition of a pattern of chemical functional groups including amino groups, carboxy groups, oxo groups and thiol groups, that can be used to covalently attach microspheres, which generally also contain corresponding reactive functional groups; the addition of a pattern of adhesive that can be used to bind the microspheres (either by prior chemical functionalization for the addition of the adhesive or direct addition of the adhesive); the addition of a pattern of charged groups (similar to the chemical functionalities) for the electrostatic attachment of the microspheres, i.e.
  • microspheres comprise charged groups opposite to the sites; the addition of a pattern of chemical functional groups that renders the sites differentially hydrophobic or hydrophilic, such that the addition of similarly hydrophobic or hydrophilic microspheres under suitable experimental conditions will result in association of the microspheres to the sites on the basis of hydroaffinity.
  • hydrophobic sites with hydrophobic beads in an aqueous system, drives the association of the beads preferentially onto the sites.
  • pattern in this sense includes the use of a uniform treatment of the surface to allow attachment of the beads at discrete sites, as well as treatment of the surface resulting in discrete sites. As will be appreciated by those in the art, this may be accomplished in a variety of ways.
  • compositions of the invention further comprise a population of microspheres.
  • population herein is meant a plurality of beads as outlined above for arrays. Within the population are separate subpopulations, which can be a single microsphere or multiple identical microspheres. That is, in some embodiments, as is more fully outlined below, the array may contain only a single bead for each bioactive agent; preferred embodiments utilize a plurality of beads of each type.
  • microspheres or “beads” or “particles” or grammatical equivalents herein is meant small discrete particles.
  • the composition of the beads will vary, depending on the class of bioactive agent and the method of synthesis. Suitable bead compositions include those used in peptide, nucleic acid and organic moiety synthesis, including, but not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphited, titanium dioxide, latex or cross-linked dextrans such as Sepharose, cellulose, nylon, cross-linked micelles and teflon may all be used. “Microsphere Detection Guide” from Bangs Laboratories, Fishers IN is a helpful guide.
  • the beads need not be spherical; irregular particles may be used.
  • the beads may be porous, thus increasing the surface area of the bead available for assay.
  • the bead sizes range from nanometers, i.e. 100 nm, to millimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200 microns being preferred, and from about 0.5 to about 5 micron being particularly preferred, although in some embodiments smaller beads may be used.
  • a key component of the invention is the use of a substrate/bead pairing that allows the association or attachment of the beads at discrete sites on the surface of the substrate, such that the beads do not move during the course of the assay.
  • Each microsphere comprises a capture probe although as will be appreciated by those in the art, there may be some microspheres which do not contain a capture probe, depending the on the synthetic methods.
  • capture probe or “capture nucleic acid” herein is meant a probe for the direct or indirect binding of the target sequence to a bead.
  • nucleic acid or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together, as described above.
  • each bead comprises a single type of capture probes, although a plurality of individual probes are preferably attached to each bead.
  • preferred embodiments utilize more than one microsphere containing a unique capture probe; that is, there is redundancy built into the system by the use of subpopulations of microspheres, each microsphere in the subpopulation containing the same probe.
  • the probes may either be synthesized directly on the beads, or they may be made and then attached after synthesis.
  • linkers are used to attach the probes to the beads, to allow both good attachment, sufficient flexibility to allow good interaction with the target sequence, and to avoid undesirable binding reactions.
  • the probes are synthesized directly on the beads.
  • many classes of chemical compounds are currently synthesized on solid supports, such as peptides, organic moieties, and nucleic acids. It is a relatively straightforward matter to adjust the current synthetic techniques to use beads.
  • the probes are synthesized first, and then covalently attached to the beads. As will be appreciated by those in the art, this will be done depending on the composition of the bioactive agents and the beads.
  • the functionalization of solid support surfaces such as certain polymers with chemically reactive groups such as thiols, amines, carboxyls, etc. is generally known in the art. Accordingly, “blank” microspheres may be used that have surface chemistries that facilitate the attachment of the desired functionality by the user.
  • Some examples of these surface chemistries for blank microspheres include, but are not limited to, amino groups including aliphatic and aromatic amines, carboxylic acids, aldehydes, amides, chloromethyl groups, hydrazide, hydroxyl groups, sulfonates and sulfates.
  • the beads may additionally comprise an optical signature, that can be used to identify the bioactive agent; see for example U.S. Ser. Nos. 08/818,199 and 09/151,877, and PCT US98/05025, all of which are expressly incorporated herein by reference.
  • the microspheres may additionally comprise identifier binding ligands for use in certain decoding systems.
  • identifier binding ligands or “IBLs” herein is meant a compound that will specifically bind a corresponding decoder binding ligand (DBL) to facilitate the elucidation of the identity of the bioactive agent attached to the bead. That is, the IBL and the corresponding DBL form a binding partner pair.
  • specifically bind herein is meant that the IBL binds its DBL with specificity sufficient to differentiate between the corresponding DBL and other DBLs (that is, DBLs for other IBLs), or other components or contaminants of the system.
  • the binding should be sufficient to remain bound under the conditions of the decoding step, including wash steps to remove non-specific binding.
  • the dissociation constants of the IBL to its DBL will be less than about 10 ⁇ 4 -10 ⁇ 6 M ⁇ 1 , with less than about 10 ⁇ 5 to 10 ⁇ 9 M ⁇ 1 being preferred and less than about 10 ⁇ 7 -10 ⁇ 9 M ⁇ 1 being particularly preferred.
  • IBL-DBL binding pairs are known or can be readily found using known techniques.
  • the DBLs include proteins (particularly including antibodies or fragments thereof (FAbs, etc.)) or small molecules, or vice versa (the IBL is an antibody and the DBL is a protein).
  • protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
  • the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids.
  • homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention.
  • the side chains may be in either the (R) or the (S) configuration.
  • the amino acids are in the (S) or L-configuration.
  • non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
  • Metal ion-metal ion ligands or chelators pairs are also useful.
  • Antigen-antibody pairs, enzymes and substrates or inhibitors, other protein-protein interacting pairs, receptor-ligands, complementary nucleic acids, and carbohydrates and their binding partners are also suitable binding pairs.
  • nucleic acid-nucleic acid binding proteins pairs are also useful. Similarly, as is generally described in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and related patents, hereby incorporated by reference, nucleic acid “aptomers” can be developed for binding to virtually any target; such a aptomer-target pair can be used as the IBL-DBL pair. Similarly, there is a wide body of literature relating to the development of binding pairs based on combinatorial chemistry methods.
  • the IBL is a molecule whose color or luminescence properties change in the presence of a selectively-binding DBL.
  • the IBL may be a fluorescent pH indicator whose emission intensity changes with pH.
  • the IBL may be a fluorescent ion indicator, whose emission properties change with ion concentration.
  • the IBL is a molecule whose color or luminescence properties change in the presence of various solvents.
  • the IBL may be a fluorescent molecule such as an ethidium salt whose fluorescence intensity increases in hydrophobic environments.
  • the IBL may be a derivative of fluorescein whose color changes between aqueous and nonpolar solvents.
  • the DBL may be attached to a bead, i.e. a “decoder bead”, that may carry a label such as a fluorophore.
  • the IBL-DBL pair comprise substantially complementary single-stranded nucleic acids.
  • the binding ligands can be referred to as “identifier probes” and “decoder probes”.
  • the identifier and decoder probes range from about 4 basepairs in length to about 1000, with from about 6 to about 100 being preferred, and from about 8 to about 40 being particularly preferred. What is important is that the probes are long enough to be specific, i.e. to distinguish between different IBL-DBL pairs, yet short enough to allow both a) dissociation, if necessary, under suitable experimental conditions, and b) efficient hybridization.
  • the IBLs do not bind to DBLs. Rather, the IBLs are used as identifier moieties (“IMs”) that are identified directly, for example through the use of mass spectroscopy.
  • IMs identifier moieties
  • the microspheres may contain an optical signature. That is, as outlined in U.S. Ser. Nos. 08/818,199 and 09/151,877, previous work had each subpopulation of microspheres comprising a unique optical signature or optical tag that is used to identify the unique capture probe of that subpopulation of microspheres; that is, decoding utilizes optical properties of the beads such that a bead comprising the unique optical signature may be distinguished from beads at other locations with different optical signatures. Thus the previous work assigned each probe a unique optical signature such that any microspheres comprising that probe are identifiable on the basis of the signature. These optical signatures comprised dyes, usually chromophores or fluorophores, that were entrapped or attached to the beads themselves. Diversity of optical signatures utilized different fluorochromes, different ratios of mixtures of fluorochromes, and different concentrations (intensities) of fluorochromes.
  • the present invention does not rely solely on the use of optical properties to decode the arrays, as will be appreciated by those in the art, it is possible in some embodiments to utilize optical signatures as an additional coding method, in conjunction with the present system.
  • the size of the array may be effectively increased while using a single set of decoding moieties in several ways, one of which is the use of optical signatures one some beads.
  • the use of two populations of beads, one with an optical signature and one without allows the effective doubling of the array size.
  • the use of multiple optical signatures similarly increases the possible size of the array.
  • each subpopulation of beads comprises a plurality of different IBLs.
  • the number of possible unique codes is substantially increased. That is, by using one unique IBL per probe, the size of the array will be the number of unique IBLs (assuming no “reuse” occurs, as outlined below).
  • the size of the array can be increased to 2 n , when the presence or absence of each IBL is used as the indicator. For example, the assignment of 10 IBLs per bead generates a 10 bit binary code, where each bit can be designated as “1” (IBL is present) or “0” (IBL is absent).
  • a 10 bit binary code has 2 10 possible variants.
  • the size of the array may be further increased if another parameter is included such as concentration or intensity; thus for example, if two different concentrations of the IBL are used, then the array size increases as 3 n .
  • each individual probe in the array is assigned a combination of IBLs, which can be added to the beads prior to the addition of the probe, after, or during the synthesis of the probe, i.e. simultaneous addition of IBLs and probes.
  • the combination of different IBLs can be used to elucidate the sequence of the probe.
  • adenosine can be represented by the presence of both IBL1 and IBL2; thymidine can be represented by the presence of IBL1 but not IBL2, cytosine can be represented by the presence of IBL2 but not IBL1, and guanosine can be represented by the absence of both.
  • the second position of the nucleic acid can be done in a similar manner using IBL3 and IBL4; thus, the presence of IBL1, IBL2, IBL3 and IBL4 gives a sequence of AA; IBL1, IBL2, and IBL3 shows the sequence AT; IBL1, IBL3 and IBL4 gives the sequence TA, etc.
  • the third position utilizes IBL5 and IBL6, etc. In this way, the use of 20 different identifiers can yield a unique code for every possible 10-mer.
  • the use of different concentrations or densities of IBLs allows a “reuse” of sorts. If, for example, the bead comprising a first agent has a 1 ⁇ concentration of IBL, and a second bead comprising a second agent has a 10 ⁇ concentration of IBL, using saturating concentrations of the corresponding labelled DBL allows the user to distinguish between the two beads.
  • the microspheres comprising the probes and the unique tags are generated, they are added to the substrate to form an array.
  • the methods of making the arrays and of decoding the arrays is done to maximize the number of different candidate agents that can be uniquely encoded.
  • the compositions of the invention may be made in a variety of ways.
  • the arrays are made by adding a solution or slurry comprising the beads to a surface containing the sites for attachment of the beads. This may be done in a variety of buffers, including aqueous and organic solvents, and mixtures. The solvent can evaporate, and excess beads removed.
  • a novel method of loading the beads onto the array comprises exposing the array to a solution of particles (including microspheres and cells) and then applying energy, e.g. agitating or vibrating the mixture. This results in an array comprising more tightly associated particles, as the agitation is done with sufficient energy to cause weakly-associated beads to fall off (or out, in the case of wells). These sites are then available to bind a different bead. In this way, beads that exhibit a high affinity for the sites are selected.
  • arrays made in this way have two main advantages as compared to a more static loading: first of all, a higher percentage of the sites can be filled easily, and secondly, the arrays thus loaded show a substantial decrease in bead loss during assays.
  • these methods are used to generate arrays that have at least about 50% of the sites filled, with at least about 75% being preferred, and at least about 90% being particularly preferred.
  • arrays generated in this manner preferably lose less than about 20% of the beads during an assay, with less than about 10% being preferred and less than about 5% being particularly preferred.
  • the substrate comprising the surface with the discrete sites is immersed into a solution comprising the particles (beads, cells, etc.).
  • the surface may comprise wells, as is described herein, or other types of sites on a patterned surface such that there is a differential affinity for the sites. This differential affinity results in a competitive process, such that particles that will associate more tightly are selected.
  • the entire surface to be “loaded” with beads is in fluid contact with the solution.
  • This solution is generally a slurry ranging from about 10,000:1 beads:solution (vol:vol) to 1:1.
  • the solution can comprise any number of reagents, including aqueous buffers, organic solvents, salts, other reagent components, etc.
  • the solution preferably comprises an excess of beads; that is, there are more beads than sites on the array. Preferred embodiments utilize two-fold to billion-fold excess of beads.
  • the immersion can mimic the assay conditions; for example, if the array is to be “dipped” from above into a microtiter plate comprising samples, this configuration can be repeated for the loading, thus minimizing the beads that are likely to fall out due to gravity.
  • the substrate, the solution, or both are subjected to a competitive process, whereby the particles with lower affinity can be disassociated from the substrate and replaced by particles exhibiting a higher affinity to the site.
  • This competitive process is done by the introduction of energy, in the form of heat, sonication, stirring or mixing, vibrating or agitating the solution or substrate, or both.
  • a preferred embodiment utilizes agitation or vibration.
  • the amount of manipulation of the substrate is minimized to prevent damage to the array; thus, preferred embodiments utilize the agitation of the solution rather than the array, although either will work.
  • this agitation can take on any number of forms, with a preferred embodiment utilizing microtiter plates comprising bead solutions being agitated using microtiter plate shakers.
  • the agitation proceeds for a period of time sufficient to load the array to a desired fill. Depending on the size and concentration of the beads and the size of the array, this time may range from about 1 second to days, with from about 1 minute to about 24 hours being preferred.
  • sites of an array may comprise a bead; that is, there may be some sites on the substrate surface which are empty. In addition, there may be some sites that contain more than one bead, although this is not preferred.
  • the beads in a non-random or ordered way.
  • photoactivatible attachment linkers or photoactivatible adhesives or masks selected sites on the array may be sequentially rendered suitable for attachment, such that defined populations of beads are laid down.
  • the arrays of the present invention are constructed such that information about the identity of the candidate agent is built into the array, such that the random deposition of the beads in the fiber wells can be “decoded” to allow identification of the candidate agent at all positions. This may be done in a variety of ways, and either before, during or after the use of the array to detect target molecules.
  • the array is made, it is “decoded” in order to identify the location of one or more of the probes, i.e. each subpopulation of beads, on the substrate surface.
  • a selective decoding system is used. In this case, only those microspheres exhibiting a change in the optical signal as a result of the binding of a target analyte are decoded. This is commonly done when the number of “hits”, i.e. the number of sites to decode, is generally low. That is, the array is first scanned under experimental conditions in the absence of the target analytes. The sample containing the target analytes is added, and only those locations exhibiting a change in the optical signal are decoded.
  • the beads at either the positive or negative signal locations may be either selectively tagged or released from the array (for example through the use of photocleavable linkers), and subsequently sorted or enriched in a fluorescence-activated cell sorter (FACS). That is, either all the negative beads are released, and then the positive beads are either released or analyzed in situ, or alternatively all the positives are released and analyzed.
  • the labels may comprise halogenated aromatic compounds, and detection of the label is done using for example gas chromatography, chemical tags, isotopic tags mass spectral tags.
  • each bead contains a fluorescent dye.
  • the identification may occur through the use of identifier moieties (“IMs”), which are similar to IBLs but need not necessarily bind to DBLs. That is, rather than elucidate the structure of the capture probe directly, the composition of the IMs may serve as the identifier.
  • IMs identifier moieties
  • a specific combination of IMs can serve to code the bead, and be used to identify the agent on the bead upon release from the bead followed by subsequent analysis, for example using a gas chromatograph or mass spectroscope.
  • each bead comprises a non-fluorescent precursor to a fluorescent dye.
  • photocleavable protecting groups such as certain ortho-nitrobenzyl groups
  • photoactivation of the fluorochrome can be done. After the assay, light is shown down again either the “positive” or the “negative” fibers, to distinquish these populations.
  • the illuminated precursors are then chemically converted to a fluorescent dye. All the beads are then released from the array, with sorting, to form populations of fluorescent and non-fluorescent beads (either the positives and the negatives or vice versa).
  • the sites of attachment of the beads include a photopolymerizable reagent, or the photopolymerizable agent is added to the assembled array. After the test assay is run, light is shown down again either the “positive” or the “negative” fibers, to distinquish these populations. As a result of the irradiation, either all the positives or all the negatives are polymerized and trapped or bound to the sites, while the other population of beads can be released from the array.
  • the location of every capture probe is determined using decoder binding ligands (DBLs).
  • DBLs are binding ligands that will either bind to identifier binding ligands, if present, or to the capture probes themselves.
  • the DBL binds to the IBL.
  • the capture probes are single-stranded nucleic acids and the DBL is a substantially complementary single-stranded nucleic acid that binds (hybridizes) to the capture probe, termed a decoder probe herein.
  • a decoder probe that is substantially complementary to each candidate probe is made and used to decode the array.
  • the candidate probes and the decoder probes should be of sufficient length (and the decoding step run under suitable conditions) to allow specificity; i.e. each candidate probe binds to its corresponding decoder probe with sufficient specificity to allow the distinction of each candidate probe.
  • the DBLs are either directly or indirectly labeled.
  • label herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound.
  • labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic, electrical, thermal; and c) colored or luminescent dyes; although labels include enzymes and particles such as magnetic particles as well.
  • Preferred labels include luminescent labels.
  • the DBL is directly labeled, that is, the DBL comprises a label.
  • the DBL is indirectly labeled; that is, a labeling binding ligand (LBL) that will bind to the DBL is used.
  • LBL labeling binding ligand
  • the labeling binding ligand-DBL pair can be as described above for IBL-DBL pairs.
  • the identification of the location of the individual beads is done using one or more decoding steps comprising a binding between the labeled DBL and either the IBL or the capture probe (i.e. a hybridization between the capture probe and the decoder probe).
  • the DBLs can be removed and the array can be used; however, in some circumstances, for example when the DBL binds to an IBL and not to the capture probe, the removal of the DBL is not required (although it may be desirable in some circumstances).
  • decoding may be done either before the array is used to in an assay, during the assay, or after the assay.
  • each DBL is labeled with a unique label, such that the number of unique tags is equal to or greater than the number of capture probe (although in some cases, “reuse” of the unique labels can be done, as described herein; similarly, minor variants of candidate probes can share the same decoder, if the variants are encoded in another dimension, i.e. in the bead size or label).
  • a DBL is made that will specifically bind to it and contains a unique tag, for example one or more fluorochromes.
  • the location of each DBL can be elucidated. This allows the identification of the location of each capture probe; the random array has been decoded.
  • the DBLs can then be removed, if necessary, and the target sample applied.
  • the number of unique labels is less than the number of unique bioactive agents, and thus a sequential series of decoding steps are used.
  • decoder probes are divided into n sets for decoding. The number of sets corresponds to the number of unique tags. Each decoder probe is labeled in n separate reactions with n distinct tags. All the decoder probes share the same n tags. The decoder probes are pooled so that each pool contains only one of the n tag versions of each decoder, and no two decoder probes have the same sequence of tags across all the pools.
  • the number of pools required for this to be true is determined by the number of decoder probes and the n.
  • Hybridization of each pool to the array generates a signal at every address.
  • the sequential hybridization of each pool in turn will generate a unique, sequence-specific code for each candidate probe. This identifies the candidate probe at each address in the array. For example, if four tags are used, then 4 ⁇ n sequential hybridizations can ideally distinguish 4 n sequences, although in some cases more steps may be required.
  • the hybrids are denatured and the decoder probes removed, so that the probes are rendered single-stranded for the next hybridization (although it is also possible to hybridize limiting amounts of target so that the available probe is not saturated. Sequential hybridizations can be carried out and analyzed by subtracting pre-existing signal from the previous hybridization).
  • Decoder probes 1-16 are made that correspond to the probes on the beads.
  • the first step is to label decoder probes 1-4 with tag A, decoder probes 5-8 with tag B, decoder probes 9-12 with tag C, and decoder probes 13-16 with tag D.
  • the probes are mixed and the pool is contacted with the array containing the beads with the attached candidate probes.
  • the location of each tag (and thus each decoder and candidate probe pair) is then determined.
  • the first set of decoder probes are then removed.
  • decoder probes 1, 5, 9 and 13 are labeled with tag A
  • decoder probes 2, 6, 10 and 14 are labeled with tag B
  • decoder probes 3, 7, 11 and 15 are labeled with tag C
  • decoder probes 4, 8, 12 and 16 are labeled with tag D.
  • those beads that contained tag A in both decoding steps contain candidate probe 1; tag A in the first decoding step and tag B in the second decoding step contain candidate probe 2; tag A in the first decoding step and tag C in the second step contain candidate probe 3; etc.
  • the decoder probes are labeled in situ; that is, they need not be labeled prior to the decoding reaction.
  • the incoming decoder probe is shorter than the candidate probe, creating a 5′ “overhang” on the decoding probe.
  • the addition of labeled ddNTPs (each labeled with a unique tag) and a polymerase will allow the addition of the tags in a sequence specific manner, thus creating a sequence-specific pattern of signals.
  • other modifications can be done, including ligation, etc.
  • the size of the array will be set by the number of unique decoding binding ligands, it is possible to “reuse” a set of unique DBLs to allow for a greater number of test sites. This may be done in several ways; for example, by using some subpopulations that comprise optical signatures. Similarly, the use of a positional coding scheme within an array; different sub-bundles may reuse the set of DBLs. Similarly, one embodiment utilizes bead size as a coding modality, thus allowing the reuse of the set of unique DBLs for each bead size. Alternatively, sequential partial loading of arrays with beads can also allow the reuse of DBLs. Furthermore, “code sharing” can occur as well.
  • the DBLs may be reused by having some subpopulations of beads comprise optical signatures.
  • the optical signature is generally a mixture of reporter dyes, preferably fluorescent.
  • the composition of the mixture i.e. the ratio of one dye to another
  • concentration of the dye leading to differences in signal intensity
  • matrices of unique optical signatures may be generated. This may be done by covalently attaching the dyes to the surface of the beads, or alternatively, by entrapping the dye within the bead.
  • the dyes may be chromophores or phosphors but are preferably fluorescent dyes, which due to their strong signals provide a good signal-to-noise ratio for decoding.
  • Suitable dyes for use in the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, and others described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference.
  • fluorescent lanthanide complexes including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueTM, Texas Red, and others described in the 6th Edition of the Molecular Probes Handbook by Richard P.
  • the encoding can be accomplished in a ratio of at least two dyes, although more encoding dimensions may be added in the size of the beads, for example.
  • the labels are distinguishable from one another; thus two different labels may comprise different molecules (i.e. two different fluors) or, alternatively, one label at two different concentrations or intensity.
  • the dyes are covalently attached to the surface of the beads. This may be done as is generally outlined for the attachment of the capture probe, using functional groups on the surface of the beads. As will be appreciated by those in the art, these attachments are done to minimize the effect on the dye.
  • the dyes are non-covalently associated with the beads, generally by entrapping the dyes in the pores of the beads.
  • a spatial or positional coding system is done.
  • each subarray is an “area code”, that can have the same tags (i.e. telephone numbers) of other subarrays, that are separated by virtue of the location of the subarray.
  • tags i.e. telephone numbers
  • the same unique tags can be reused from bundle to bundle.
  • the use of 50 unique tags in combination with 100 different subarrays can form an array of 5000 different bioactive agents.
  • additional encoding parameters can be added, such as microsphere size.
  • the use of different size beads may also allow the reuse of sets of DBLs; that is, it is possible to use microspheres of different sizes to expand the encoding dimensions of the microspheres.
  • Optical fiber arrays can be fabricated containing pixels with different fiber diameters or cross-sections; alternatively, two or more fiber optic bundles, each with different cross-sections of the individual fibers, can be added together to form a larger bundle; or, fiber optic bundles with fiber of the same size cross-sections can be used, but just with different sized beads.
  • the largest wells can be filled with the largest microspheres and then moving onto progressively smaller microspheres in the smaller wells until all size wells are then filled.
  • the same dye ratio could be used to encode microspheres of different sizes thereby expanding the number of different oligonucleotide sequences or chemical functionalities present in the array.
  • the coding and decoding is accomplished by sequential loading of the microspheres into the array.
  • the optical signatures can be “reused”.
  • the library of microspheres each comprising a different bioactive agent or the subpopulations each comprise a different bioactive agent
  • the library of microspheres each comprising a different bioactive agent is divided into a plurality of sublibraries; for example, depending on the size of the desired array and the number of unique tags, 10 sublibraries each comprising roughly 10% of the total library may be made, with each sublibrary comprising roughly the same unique tags.
  • the first sublibrary is added to the fiber optic bundle comprising the wells, and the location of each bioactive agent is determined, generally through the use of DBLs.
  • the second sublibrary is then added, and the location of each bioactive agent is again determined.
  • the signal in this case will comprise the signal from the “first” DBL and the “second” DBL; by comparing the two matrices the location of each bead in each sublibrary can be determined. Similarly, adding the third, fourth, etc. sublibraries sequentially will allow the array to be filled.
  • codes can be “shared” in several ways.
  • a single code i.e. IBL/DBL pair
  • two nucleic acid probes used in an mRNA quantitation assay can share the same code if the ranges of their hybridization signal intensities do not overlap. This can occur, for example, when one of the target sequences is always present at a much higher concentration than the other. Alternatively, the two target sequences might always be present at a similar concentration, but differ in hybridization efficiency.
  • a single code can be assigned to multiple agents if the agents are functionally equivalent.
  • the probes are functionally equivalent, even though they may differ in sequence.
  • all probes for different members of a class such as kinases or G-protein coupled receptors could share a code.
  • an array of this type could be used to detect homologs of known genes.
  • each gene is represented by a heterologous set of probes, hybridizing to different regions of the gene (and therefore differing in sequence). The set of probes share a common code.
  • a homolog If a homolog is present, it might hybridize to some but not all of the probes. The level of homology might be indicated by the fraction of probes hybridizing, as well as the average hybridization intensity. Similarly, multiple antibodies to the same protein could all share the same code.
  • sensor redundancy utilizes of a plurality of sensor elements such as beads comprising identical binding ligands such as probes.
  • Target redundancy utilizes sensor elements with different probes to the same target: one probe may span the first 25 bases of the target, a second probe may span the second 25 bases of the target, etc.
  • sensor redundancy is used.
  • a plurality of sensor elements e.g. beads, comprising identical bioactive agents are used. That is, each subpopulation comprises a plurality of beads comprising identical bioactive agents (e.g. binding ligands).
  • bioactive agents e.g. binding ligands.
  • the optical signal from each sensor element can be combined and any number of statistical analyses run, as outlined below. This can be done for a variety of reasons. For example, in time varying measurements, redundancy can significantly reduce the noise in the system. For non-time based measurements, redundancy can significantly increase the confidence of the data.
  • a plurality of identical sensor elements are used.
  • the number of identical sensor elements will vary with the application and use of the sensor array. In general, anywhere from 2 to thousands may be used, with from 2 to 100 being preferred, 2 to 50 being particularly preferred and from 5 to 20 being especially preferred. In general, preliminary results indicate that roughly 10 beads gives a sufficient advantage, although for some applications, more identical sensor elements can be used.
  • optical response signals from a plurality of sensor beads within each bead subpopulation can be manipulated and analyzed in a wide variety of ways, including baseline adjustment, averaging, standard deviation analysis, distribution and cluster analysis, confidence interval analysis, mean testing, etc.
  • the first manipulation of the optical response signals is an optional baseline adjustment.
  • the standardized optical responses are adjusted to start at a value of 0.0 by subtracting the integer 1.0 from all data points. Doing this allows the baseline-loop data to remain at zero even when summed together and the random response signal noise is canceled out.
  • the fluid pulse-loop temporal region frequently exhibits a characteristic change in response, either positive, negative or neutral, prior to the sample pulse and often requires a baseline adjustment to overcome noise associated with drift in the first few data points due to charge buildup in the CCD camera.
  • the baseline from the first data point for each bead sensor is subtracted from all the response data for the same bead. If drift is observed, the average baseline from the first ten data points for each bead sensor is substracted from the all the response data for the same bead.
  • this baseline adjustment when multiple bead responses are added together they can be amplified while the baseline remains at zero. Since all beads respond at the same time to the sample (e.g. the sample pulse), they all see the pulse at the exact same time and there is no registering or adjusting needed for overlaying their responses.
  • other types of baseline adjustment may be done, depending on the requirements and output of the system used.
  • signal summing is done by simply adding the intensity values of all responses at each time point, generating a new temporal response comprised of the sum of all bead responses. These values can be baseline-adjusted or raw. As for all the analyses described herein, signal summing can be performed in real time or during post-data acquisition data reduction and analysis. In one embodiment, signal summing is performed with a commercial spreadsheet program (Excel, Microsoft, Redmond, Wash.) after optical response data is collected.
  • cummulative response data is generated by simply adding all data points in successive time intervals. This final column, comprised of the sum of all data points at a particular time interval, may then be compared or plotted with the individual bead responses to determine the extent of signal enhancement or improved signal-to-noise ratios.
  • the mean of the subpopulation i.e. the plurality of identical beads
  • Equation 1 the mean of the subpopulation
  • the subpopulation may be redefined to exclude some beads if necessary (for example for obvious outliers, as discussed below).
  • the standard deviation of the subpopulation can be determined, generally using Equation 2 (for the entire subpopulation) and Equation 3 (for less than the entire subpopulation):
  • Equation 2 ⁇ ⁇ ( x i - ⁇ ) 2 n Equation ⁇ ⁇ 2
  • Equation 3 s ⁇ ( x i - x _ ) 2 n - 1 Equation ⁇ ⁇ 3
  • the subpopulation may be redefined to exclude some beads if necessary (for example for obvious outliers, as discussed below).
  • statistical analyses are done to evaluate whether a particular data point has statistical validity within a subpopulation by using techniques including, but not limited to, t distribution and cluster analysis. This may be done to statistically discard outliers that may otherwise skew the result and increase the signal-to-noise ratio of any particular experiment. This may be done using Equation 4:
  • Equation 4 t x _ - ⁇ s / n Equation ⁇ ⁇ 4
  • the quality of the data is evaluated using confidence intervals, as is known in the art. Confidence intervals can be used to facilitate more comprehensive data processing to measure the statistical validity of a result.
  • statistical parameters of a subpopulation of beads are used to do hypothesis testing.
  • tests concerning means also called mean testing.
  • statistical evaluation is done to determine whether two subpopulations are different. For example, one sample could be compared with another sample for each subpopulation within an array to determine if the variation is statistically significant.
  • mean testing can also be used to differentiate two different assays that share the same code. If the two assays give results that are statistically distinct from each other, then the subpopulations that share a common code can be distinguished from each other on the basis of the assay and the mean test, shown below in Equation 5:
  • analyzing the distribution of individual members of a subpopulation of sensor elements may be done. For example, a subpopulation distribution can be evaluated to determine whether the distribution is binomial, Poisson, hypergeometric, etc.
  • a preferred embodiment utilizes a plurality of sensor elements that are directed to a single target analyte but yet are not identical.
  • a single target nucleic acid analyte may have two or more sensor elements each comprising a different probe. This adds a level of confidence as non-specific binding interactions can be statistically minimized.
  • the redundant nucleic acid probes may be overlapping, adjacent, or spatially separated. However, it is preferred that two probes do not compete for a single binding site, so adjacent or separated probes are preferred.
  • bioactive agent binding agents that bind to different parts of the target.
  • preferred embodiments utilize antibodies (or antibody fragments) as bioactive agents for the binding of target proteins.
  • a plurality of different sensor elements may be used, with from about 2 to about 20 being preferred, and from about 2 to about 10 being especially preferred, and from 2 to about 5 being particularly preferred, including 2, 3, 4 or 5. However, as above, more may also be used, depending on the application.
  • any number of statistical analyses may be run on the data from target redundant sensors.
  • Bead summing One benefit of the sensor element summing (referred to herein as “bead summing” when beads are used), is the increase in sensitivity that can occur.
  • compositions of the invention find use in a number of applications.
  • the probes are used in genetic diagnosis.
  • probes can be made using the techniques disclosed herein to detect target sequences such as the gene for nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which is a gene associated with a variety of cancers, the Apo E4 gene that indicates a greater risk of Alzheimer's disease, allowing for easy presymptomatic screening of patients, mutations in the cystic fibrosis gene, cytochrome p450s or any of the others well known in the art.
  • viral and bacterial detection is done using the complexes of the invention.
  • probes are designed to detect target sequences from a variety of bacteria and viruses.
  • current blood-screening techniques rely on the detection of anti-HIV antibodies.
  • the methods disclosed herein allow for direct screening of clinical samples to detect HIV nucleic acid sequences, particularly highly conserved HIV sequences. In addition, this allows direct monitoring of circulating virus within a patient as an improved method of assessing the efficacy of anti-viral therapies.
  • viruses associated with leukemia, HTLV-I and HTLV-II may be detected in this way.
  • Bacterial infections such as tuberculosis, chlamydia and other sexually transmitted diseases, may also be detected.
  • the nucleic acids of the invention find use as probes for toxic bacteria in the screening of water and food samples.
  • samples may be treated to lyse the bacteria to release its nucleic acid, and then probes designed to recognize bacterial strains, including, but not limited to, such pathogenic strains as, Salmonella, Campylobacter, Vibrio cholerae, Leishmania, enterotoxic strains of E. coli, and Legionnaire's disease bacteria.
  • bioremediation strategies may be evaluated using the compositions of the invention.
  • the probes are used for forensic “DNA fingerprinting” to match crime-scene DNA against samples taken from victims and suspects.
  • the probes in an array are used for sequencing by hybridization.
  • the present invention also finds use as a methodology for the detection of mutations or mismatches in target nucleic acid sequences.
  • recent focus has been on the analysis of the relationship between genetic variation and phenotype by making use of polymorphic DNA markers.
  • Previous work utilized short tandem repeats (STRs) as polymorphic positional markers; however, recent focus is on the use of single nucleotide polymorphisms (SNPs), which occur at an average frequency of more than 1 per kilobase in human genomic DNA.
  • SNPs single nucleotide polymorphisms
  • a change in optical signal occurs as a result of the binding of a target analyte that is labeled, either directly or indirectly, with a detectable label, preferably an optical label such as a fluorochrome.
  • a detectable label preferably an optical label such as a fluorochrome.
  • a nucleic acid target analyte when used, it may be either directly labeled with a fluor, or indirectly, for example through the use of a labeled antibody.
  • nucleic acids are easily labeled with fluorochromes, as outlined herein. For example during PCR amplification as is known in the art.
  • a hybridization indicator may be used as the label.
  • Hybridization indicators preferentially associate with double stranded nucleic acid, usually reversibly.
  • Hybridization indicators include intercalators and minor and/or major groove binding moieties.
  • intercalators may be used; since intercalation generally only occurs in the presence of double stranded nucleic acid, only in the presence of target hybridization will the label light up. Thus, upon binding of the target analyte to a bioactive agent, there is a new optical signal generated at that site, which then may be detected.
  • a change in the optical signature may be the basis of the optical signal.
  • the interaction of some chemical target analytes with some fluorescent dyes on the beads may alter the optical signature, thus generating a different optical signal.
  • the assays may be run under a variety of experimental conditions, as will be appreciated by those in the art.
  • a variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions.
  • reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used.
  • the mixture of components may be added in any order that provides for the requisite binding.
  • Various blocking and washing steps may be utilized as is known in the art.
  • the methods of the invention are useful in array quality control. Prior to this invention, no methods have been described that provide a positive test of the performance of every probe on every array. Decoding of the array not only provides this test, it also does so by making use of the data generated during the decoding process itself. Therefore, no additional experimental work is required.
  • the invention requires only a set of data analysis algorithms that can be encoded in software.
  • the quality control procedure can identify a wide variety of systematic and random problems in an array. For example, random specks of dust or other contaminants might cause some sensors to give an incorrect signal-this can be detected during decoding. The omission of one or more agents from multiple arrays can also be detected.
  • An advantage of this quality control procedure is that it can be implemented immediated prior to the assay itself, and is a true functional test of each individual sensor. Therefore any problems that might occur between array assembly and actual use can be detected. In applications where a very high level of confidence is required, and/or there is a significant chance of sensor failure during the experimental procedure, decoding and quality control can be conducted both before and after the actual sample analysis.
  • the arrays can be used to do reagent quality control.
  • biological macromolecules are used as reagents and must be quality controlled.
  • large sets of oligonucleotide probes may be provided as reagents. It is typically difficult to perform quality control on large numbers of different biological macromolecules.
  • the approach described here can be used to do this by treating the reagents (formulated as the DBLs) as variable instead of the arrays.
  • the methods outlined herein are used in array calibration.
  • a signal that is a linear response to the concentration of the target analyte, or, alternatively, if non-linear, to determine a relationship between concentration and signal, so that the concentration of the target analyte can be estimated.
  • the present invention provides methods of creating calibration curves in parallel for multiple beads in an array.
  • the calibration curves can be created under conditions that simulate the complexity of the sample to be analyzed. Each curve can be constructed independently of the others (e.g. for a different range of concentrations), but at the same time as all the other curves for the array.
  • the sequential decoding scheme is implemented with different concentrations being used as the code “labels”, rather than different fluorophores.
  • concentrations being used as the code “labels”, rather than different fluorophores.
  • signal as a response to concentration can be measured for each bead. This calibration can be carried out just prior to array use, so that every probe on every array is individually calibrated as needed.
  • the methods of the invention can be used in assay development as well.
  • the methods allow the identification of good and bad probes; as is understood by those in the art, some probes do not function well because they do not hybridize well, or because they cross-hybridize with more than one sequence. These problems are easily detected during decoding.
  • the ability to rapidly assess probe performance has the potential to greatly reduce the time and expense of assay development.
  • the methods of the invention are useful in quantitation in assay development.
  • Major challenges of many assays is the ability to detect differences in analyte concentrations between samples, the ability to quantitate these differences, and to measure absolute concentrations of analytes, all in the presence of a complex mixture of related analytes.
  • An example of this problem is the quantitation of a specific mRNA in the presence of total cellular mRNA.
  • One approach that has been developed as a basis of mRNA quantitation makes use of a multiple match and mismatch probe pairs (Lockhart et al., 1996), hereby incorporated by reference in its entirety. While this approach is simple, it requires relatively large numbers of probes.
  • a quantitative response to concentration is obtained by averaging the signals from a set of different probes to the gene or sequence of interest. This is necessary because only some probes respond quantitatively, and it is not possible to predict these probes with certainty. In the absence of prior knowledge, only the average response of an appropriately chosen collection of probes is quantitative. However, in the present invention, this can be applied generally to nucleic acid based assays as well as other assays. In essence, the approach is to identify the probes that respond quantitatively in a particular assay, rather than average them with other probes. This is done using the array calibration scheme outlined above, in which concentration-based codes are used.
  • Advantages of this approach include: fewer probes are needed; the accuracy of the measurement is less dependent on the number of probes used; and that the response of the sensors is known with a high level of certainty, since each and every sequence can be tested in an efficient manner. It is important to note that probes that perform well are chosen empirically, which avoids the difficulties and uncertainties of predicting probe performance, particularly in complex sequence mixtures. In contrast, in experiments described to date with ordered arrays, relatively small numbers of sequences are checked by performing quantitative spiking experiments, in which a known mRNA is added to a mixture.

Abstract

The invention relates to compositions and methods for detecting and quantifying a target nucleic acid using a variety of both signal amplification and target amplification techniques.

Description

  • The present invention is a continuation-in-part of U.S. Ser. No. 60/161,148, filed Oct. 22, 1999, which is a continuation-in-part of U.S. Ser. No. 60/135,051, filed May 20, 1999, and a continuation-in-part of U.S. Ser. No. 60/160,027, filed Oct. 22, 1999, which is a continuation-in-part of U.S. Ser. No. 60/130,089, filed Apr. 20, 1999, all of which are pending.[0001]
  • FIELD OF THE INVENTION
  • The invention relates to compositions and methods useful in the detection and quantification of a nucleic acid target using a variety of amplification techniques, including both signal amplification and target amplification. Detection proceeds through the use of a label that is associated with the amplified signal or target, either directly or indirectly, to allow optical detection of the light absorbing label using a microsphere array sensor. [0002]
  • BACKGROUND OF THE INVENTION
  • The detection of specific nucleic acids is an important tool for diagnostic medicine and molecular biology research. Gene probe assays currently play roles in identifying infectious organisms such as bacteria and viruses, in probing the expression of normal genes and identifying mutant genes such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, and for exploring homology among genes from different species. [0003]
  • Ideally, a gene probe assay should be sensitive, specific and easily automatable (for a review, see Nickerson, Current Opinion in Biotechnology 4:48-51 (1993)). The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to amplify exponentially a specific nucleic acid sequence before analysis as outlined below (for a review, see Abramson et al., Current Opinion in Biotechnology, 4:41-47 (1993)). [0004]
  • Sensitivity, i.e. detection limits, remain a significant obstacle in nucleic acid detection systems, and a variety of techniques have been developed to address this issue. Briefly, these techniques can be classified as either target amplification or signal amplification. Target amplification involves the amplification (i.e. replication) of the target sequence to be detected, resulting in a significant increase in the number of target molecules. Target amplification strategies include the polymerase chain reaction (PCR), strand displacement amplification (SDA), and nucleic acid sequence based amplification (NASBA). [0005]
  • Alternatively, rather than amplify the target, alternate techniques use the target as a template to replicate a signalling probe, allowing a small number of target molecules to result in a large number of signalling probes, that then can be detected. Signal amplification strategies include the ligase chain reaction (LCR), cycling probe technology (CPT), invasive cleavage techniques such as Invader™ technology, Q-Beta replicase (QβR) technology, and the use of “amplification probes” such as “branched DNA” that result in multiple label probes binding to a single target sequence. [0006]
  • The polymerase chain reaction (PCR) is widely used and described, and involves the use of primer extension combined with thermal cycling to amplify a target sequence; see U.S. Pat. Nos. 4,683,195 and 4,683,202, and PCR Essential Data, J. W. Wiley & sons, Ed. C. R. Newton, 1995, all of which are incorporated by reference. In addition, there are a number of variations of PCR which also find use in the invention, including “quantitative competitive PCR” or “QC-PCR”, “arbitrarily primed PCR” or “AP-PCR”, “immuno-PCR”, “Alu-PCR”, “PCR single strand conformational polymorphism” or “PCR-SSCP”, allelic PCR (see Newton et al. Nucl. Acid Res. 17:2503 91989); “reverse transcriptase PCR” or “RT-PCR”, “biotin capture PCR”, “vectorette PCR”, “panhandle PCR”, and “PCR select cDNA subtraction”, among others. [0007]
  • Strand displacement amplification (SDA) is generally described in Walker et al., in Molecular Methods for Virus Detection, Academic Press, Inc., 1995, and U.S. Pat. Nos. 5,455,166 and 5,130,238, all of which are hereby incorporated by reference. [0008]
  • Nucleic acid sequence based amplification (NASBA) is generally described in U.S. Pat. No. 5,409,818 and “Profiting from Gene-based Diagnostics”, CTB International Publishing Inc., N.J., 1996, both of which are incorporated by reference. [0009]
  • Cycling probe technology (CPT) is a nucleic acid detection system based on signal or probe amplification rather than target amplification, such as is done in polymerase chain reactions (PCR). Cycling probe technology relies on a molar excess of labeled probe which contains a scissile linkage of RNA. Upon hybridization of the probe to the target, the resulting hybrid contains a portion of RNA:DNA. This area of RNA:DNA duplex is recognized by RNAseH and the RNA is excised, resulting in cleavage of the probe. The probe now consists of two smaller sequences which may be released, thus leaving the target intact for repeated rounds of the reaction. The unreacted probe is removed and the label is then detected. CPT is generally described in U.S. Pat. Nos. 5,011,769, 5,403,711, 5,660,988, and 4,876,187, and PCT published applications WO 95/05480, WO 95/1416, and WO 95/00667, all of which are specifically incorporated herein by reference. [0010]
  • The oligonucleotide ligation assay (OLA; sometimes referred to as the ligation chain reaction (LCR)) involve the ligation of at least two smaller probes into a single long probe, using the target sequence as the template for the ligase. See generally U.S. Pat. Nos. 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO 90/01069; WO 89/12696; and WO 89/09835, all of which are incorporated by reference. [0011]
  • Invader™ technology is based on structure-specific polymerases that cleave nucleic acids in a site-specific manner. Two probes are used: an “invader” probe and a “signalling” probe, that adjacently hybridize to a target sequence with a non-complementary overlap. The enzyme cleaves at the overlap due to its recognition of the “tail”, and releases the “tail” with a label. This can then be detected. The Invader™ technology is described in U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669, all of which are hereby incorporated by reference. [0012]
  • “Rolling circle amplification” is based on extension of a circular probe that has hybridized to a target sequence. A polymerase is added that extends the probe sequence. As the circular probe has no terminus, the polymerase repeatedly extends the circular probe resulting in concatamers of the circular probe. As such, the probe is amplified. Rolling-circle amplification is generally described in Baner et al. (1998) [0013] Nuc. Acids Res. 26:5073-5078; Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88:189-193; and Lizardi et al. (1998) Nat Genet. 19:225-232, all of which are incorporated by reference in their entirety.
  • “Branched DNA” signal amplification relies on the synthesis of branched nucleic acids, containing a multiplicity of nucleic acid “arms” that function to increase the amount of label that can be put onto one probe. This technology is generally described in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporated by reference. [0014]
  • Similarily, dendrimers of nucleic acids serve to vastly increase the amount of label that can be added to a single molecule, using a similar idea but different compositions. This technology is as described in U.S. Pat. No. 5,175,270 and Nilsen et al., J. Theor. Biol. 187:273 (1997), both of which are incorporated herein by reference. [0015]
  • In each of these methods, analysis of the amplified target or amplified signal remains problematic. Accordingly, it is an object of the invention to provide compositions and methods for the detection and quantification of the products, either directly or indirectly, of nucleic acid amplification reactions. [0016]
  • SUMMARY OF THE INVENTION
  • In accordance with the objects outlined above, the present invention provides a method for detecting a first target nucleic acid sequence. In one aspect the method comprises hybridizing at least a first primer nucleic acid to the first target sequence to form a first hybridization complex, contacting the first hybridization complex with a first enzyme to form a modified first primer nucleic acid, disassociating the first hybridization complex, contacting the modified first primer nucleic acid with an array comprising a substrate with a surface comprising discrete sites and a population of microspheres comprising at least a first subpopulation comprising a first capture probe such that the first capture probe and the modified primer form an assay complex, wherein the microspheres are distributed on the surface, and detecting the presence of the modified primer nucleic acid. [0017]
  • In addition the method further comprises hybridizing at least a second primer nucleic acid to a second target sequence that is substantially complementary to the first target sequence to form a second hybridization complex, contacting the second hybridization complex with the first enzyme to form modified second primer nucleic acid, disassociating the second hybridization complex and forming a second assay complex comprising the modified second primer nucleic acid and a second capture probe on a second subpopulation. [0018]
  • In an additional aspect of the invention the primer forms a circular probe following hybridization with the target nucleic acid to form a first hybridization complex and contacting the first hybridization complex with a first enzyme comprising a ligase such that the oligonucleotide ligation assay (OLA) occurs. This is followed by adding the second enzyme, a polymerase, such that the circular probe is amplified in a rolling circle amplification (RCA) assay. [0019]
  • In an additional aspect of the invention, the first enzyme comprises a DNA polymerase and the modification is an extension of the primer such that the polymerase chain reaction (PCR) occurs. In an additional aspect of the invention the first enzyme comprises a ligase and the modification comprises a ligation of the first primer which hybridizes to a first domain of the first target sequence, to a third primer which hybridizes to a second adjacent domain of the first target sequence such that the ligase chain reaction (LCR) occurs. [0020]
  • In an additional aspect of the invention, the first primer comprises a first probe sequence, a first scissile linkage and a second probe sequence, wherein the first enzyme will cleave the scissile linkage resulting in the separation of the first and second probe sequences and the disassociation of the first hybridization complex, leaving the first target sequence intact such that the cycling probe technology (CPT) reaction occurs. [0021]
  • In addition, wherein the first enzyme is a polymerase that extends the first primer and the modified first primer comprises a first newly synthesized strand, the method further comprises the addition of a second enzyme comprising a nicking enzyme that nicks the extended first primer leaving the first target sequence intact, and extending from the nick using the polymerase, and thereby displacing the first newly synthesized strand and generating a second newly synthesized strand such that strand displacement amplification (SBA) occurs. [0022]
  • In addition, wherein the first target sequence is an RNA target sequence, the first primer nucleic acid is a DNA primer comprising an RNA polymerase promoter, the first enzyme is a reverse-transcriptase that extends the first primer to form a first newly synthesized DNA strand, the method further comprises the addition of a second enzyme comprising an RNA degrading enzyme that degrades the first target sequence, the addition of a third primer that hybridizes to the first newly synthesized DNA strand, the addition of a third enzyme comprising a DNA polymerase that extends the third primer to form a second newly synthesized DNA strand, to form a newly synthesized DNA hybrid, the addition of a fourth enzyme comprising an RNA polymerase that recognizes the RNA polymerase promoter and generates at least one newly synthesized RNA strand from the DNA hybrid, such that nucleic acid sequence-based amplification (NASBA) occurs. [0023]
  • In addition, wherein the first primer is an invader primer, the method further comprises hybridizing a signalling primer to the target sequence, the enzyme comprises a structure-specific cleaving enzyme and the modification comprises a cleavage of said signalling primer, such that the invasive cleavage reaction occurs. [0024]
  • An additional aspect of the invention is a method for detecting a target nucleic acid sequence comprising hybridizing a first primer to a first target sequence to form a first hybridization complex, contacting the first hybridization complex with a first enzyme to extend the first primer to form a first newly synthesized strand and form a nucleic acid hybrid that comprises an RNA polymerase promoter, contacting the hybrid with an RNA polymerase that recognizes the RNA polymerase promoter and generates at least one newly synthesized RNA strand, contacting the newly synthesized RNA strand with an array comprising a substrate with a surface comprising discrete sites and a population of microspheres comprising at least a first subpopulation comprising a first capture probe; such that the first capture probe and the modified primer form an assay complex; wherein the microspheres are distributed on the surface and detecting the presence of the newly synthesized RNA strand. [0025]
  • In addition, when the target nucleic acid sequence is an RNA sequence, and prior to hybridizing a first primer to a first target sequence to form a first hybridization complex, method comprises hybridizing a second primer comprising an RNA polymerase promoter sequence to the RNA sequence to form a second hybridization complex, contacting the second hybridization complex with a second enzyme to extend the second primer to form a second newly synthesized strand and form a nucleic acid hybrid; and degrading the RNA sequence to leave the second newly synthesized strand as the first target sequence. In one aspect of the invention the degrading is done by the addition of an RNA degrading enzyme. In an additional aspect of the invention the degrading is done by RNA degrading activity of reverse transcriptase. [0026]
  • In addition, when the target nucleic acid sequence is a DNA sequence, and prior to hybridizing a first primer to a first target sequence to form a first hybridization complex, the method comprises hybridizing a second primer comprising an RNA polymerase promoter sequence to the DNA sequence to form a second hybridization complex, contacting the second hybridization complex with a second enzyme to extend the second primer to form a second newly synthesized strand and form a nucleic acid hybrid, and denaturing the nucleic acid hybrid such that the second newly synthesized strand is the first target sequence. [0027]
  • An additional aspect of the invention is a kit for the detection of a first target nucleic acid sequence. The kit comprises at least a first nucleic acid primer substantially complementary to at least a first domain of the target sequence, at least a first enzyme that will modify the first nucleic acid primer, and an array comprising a substrate with a surface comprising discrete sites, and a population of microspheres comprising at least a first and a second subpopulation, wherein each subpopulation comprises a bioactive agent, wherein the microspheres are distributed on the surface. [0028]
  • In an additional aspect of the invention, is a kit for the detection of a PCR reaction wherein the first enzyme is a thermostable DNA polymerase. [0029]
  • In an additional aspect of the invention, is a kit for the detection of a LCR reaction wherein the first enzyme is a ligase and the kit comprises a first nucleic acid primer substantially complementary to a first domain of the first target sequence and a third nucleic acid primer substantially complementary to a second adjacent domain of the first target sequence. [0030]
  • In an additional aspect of the invention, is a kit for the detection of a strand displacement amplification (SDA) reaction wherein the first enzyme is a polymerase and the kit further comprises a nicking enzyme. [0031]
  • In an additional aspect of the invention, is a kit for the detection of a NASBA reaction wherein the first enzyme is a reverse transcriptase, and the kit comprises a second enzyme comprising an RNA degrading enzyme, a third primer, a third enzyme comprising a DNA polymerase and a fourth enzyme comprising an RNA polymerase. [0032]
  • In an additional aspect of the invention, is a kit for the detection of an invasive cleavage reaction wherein the first enzyme is a structure-specific cleaving enzyme, and the kit comprises a signaling primer.[0033]
  • DETAILED DESCRIPTION OF THE FIGURES
  • FIGS. 1A, 1B and [0034] 1C depict three different embodiments for attaching a target sequence to an array. The solid support 5 has microsphere 10 with capture probe 20 linked via a linker 15. FIG. 1A depicts direct attachment; the capture probe 20 hybridizes to a first portion of the target sequence 25. FIG. 1B depicts the use of a capture extender probe 30 that has a first portion that hybridizes to the capture probe 20 and a second portion that hybridizes to a first domain of the target sequence 25. FIG. 1C shows the use of an adapter sequence 35, that has been added to the target sequence, for example during an amplification reaction as outlined herein.
  • FIGS. 2A and 2B depict two preferred embodiments of SBE amplification. FIG. 2A shows [0035] extension primer 40 hybridized to the target sequence 25. Upon addition of the extension enzyme and labelled nucleotides, the extension primer is modified to form a labelled primer 41. The reaction can be repeated and then the labelled primer is added to the array as above. FIG. 2B depicts the same reaction but using adapter sequences.
  • FIGS. 3A and 3B depict two preferred embodiments of OLA amplification. FIG. 3A depicts a [0036] first ligation probe 45 and a second ligation probe 50 with a label 55. Upon addition of the ligase, the probes are ligated. The reaction can be repeated and then the ligated primer is added to the array as above. FIG. 3B depicts the same reaction but using adapter sequences.
  • FIG. 4 depicts a preferred embodiment of the invasive cleavage reaction. In this embodiment, the [0037] signaling probe 65 comprises two portions, a detection sequence 67 and a signaling portion 66. The signaling portion can serve as an adapter sequence. In addition, the signaling portion generally comprises the label 55, although as will be appreciated by those in the art, the label may be on the detection sequence as well. In addition, for optional removal of the uncleaved probes, a capture tag 60 may also be used. Upon addition of the enzyme, the structure is cleaved, releasing the signaling portion 66. The reaction can be repeated and then the signaling portion is added to the array as above.
  • FIGS. 5A and 5B depict two preferred embodiments of CPT amplification. A [0038] CPT primer 70 comprising a label 55, a first probe sequence 71 and a second probe sequence 73, separated by a scissile linkage 72, and optionally comprising a capture tag 60, is hybridized to the target sequence 25. Upon addition of the enzyme, the scissile linkage is cleaved. The reaction can be repeated and then the probe sequence comprising the label is added to the array as above. FIG. 5B depicts the same reaction but using adapter sequences.
  • FIG. 6 depicts OLA/RCA amplification using a single “padlock probe” [0039] 57. The padlock probe is hybridized with a target sequence 25. When the probe 57 is complementary to the target sequence 26, ligation of the probe termini occurs forming a circular probe 28. When the probe 57 is not complementary to the target sequence 27, ligation does not occur. Addition of polymerase and nucleotides to the circular probe results amplification of the probe 58. Cleavage of the amplified probe 58 yields fragments 59 that hybridize with an identifier probe 21 immobilized on a microsphere 10.
  • FIG. 7 depicts an alternative method of OLA/RCA. An immobilized [0040] first OLA primer 45 is hybridized with a target sequence 25 and a second OLA primer 50. Following the addition of ligase, the first and second OLA primers are ligated to form a ligated oligonucleotide 56. Following denaturation to remove the target nucleic acid, the immobilized ligated oligonucleotide is distributed on an array. An RCA probe 57 and polymerase are added to the array resulting in amplification of the circular RCA probe 58.
  • DETAILED DESCRIPTION OF THE INVENTION
  • This invention is directed to the detection (and optionally quantification) of products of nucleic acid amplification reactions, using bead arrays for detection of the amplification products. Suitable amplification methods include both target amplification and signal amplification and include, but are not limited to, polymerase chain reaction (PCR), ligation chain reaction (sometimes referred to as oligonucleotide ligase amplification OLA), cycling probe technology (CPT), strand displacement assay (SDA), transcription mediated amplification (TMA), nucleic acid sequence based amplification (NASBA), rolling circle amplification (RCA), and invasive cleavage technology. All of these methods require a primer nucleic acid (including nucleic acid analogs) that is hybridized to a target sequence to form a hybridization complex, and an enzyme is added that in some way modifies the primer to form a modified primer. For example, PCR generally requires two primers, dNTPs and a DNA polymerase; LCR requires two primers that adjacently hybridize to the target sequence and a ligase; CPT requires one cleavable primer and a cleaving enzyme; invasive cleavage requires two primers and a cleavage enzyme; etc. Thus, in general, a target nucleic acid is added to a reaction mixture that comprises the necessary amplification components, and a modified primer is formed. [0041]
  • In general, the modified primer comprises a detectable label, such as a fluorescent label, which is either incorporated by the enzyme or present on the original primer. As required, the unreacted primers are removed, in a variety of ways, as will be appreciated by those in the art and outlined herein. The hybridization complex is then disassociated, and the modified primer is detected and optionally quantitated by a microsphere array. In some cases, the newly modified primer serves as a target sequence for a secondary reaction, which then produces a number of amplified strands, which can be detected as outlined herein. [0042]
  • Accordingly, the present invention provides compositions and methods for detecting the presence or absence of target nucleic acid sequences in a sample. As will be appreciated by those in the art, the sample solution may comprise any number of things, including, but not limited to, bodily fluids (including, but not limited to, blood, urine, serum, lymph, saliva, anal and vaginal secretions, perspiration and semen, of virtually any organism, with mammalian samples being preferred and human samples being particularly preferred); environmental samples (including, but not limited to, air, agricultural, water and soil samples); biological warfare agent samples; research samples; purified samples, such as purified genomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus, genomic DNA, etc.; As will be appreciated by those in the art, virtually any experimental manipulation may have been done on the sample. [0043]
  • The present invention provides compositions and methods for detecting the presence or absence of target nucleic acid sequences in a sample. By “nucleic acid” or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 [0044] page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of labels, or to increase the stability and half-life of such molecules in physiological environments.
  • As will be appreciated by those in the art, all of these nucleic acid analogs may find use in the present invention. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occuring nucleic acids and analogs may be made. [0045]
  • Particularly preferred are peptide nucleic acids (PNA) which includes peptide nucleic acid analogs. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages. First, the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9° C. This allows for better detection of mismatches. Similarly, due to their non-ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. [0046]
  • The nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc. A preferred embodiment utilizes isocytosine and isoguanine in nucleic acids designed to be complementary to other probes, rather than target sequences, as this reduces non-specific hybridization, as is generally described in U.S. Pat. No. 5,681,702. As used herein, the term “nucleoside” includes nucleotides as well as nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides. In addition, “nucleoside” includes non-naturally occuring analog structures. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside. [0047]
  • The compositions and methods of the invention are directed to the detection of target sequences. The term “target sequence” or “target nucleic acid” or grammatical equivalents herein means a nucleic acid sequence on a single strand of nucleic acid. The target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others. As is outlined herein, the target sequence may be a target sequence from a sample, or a secondary target such as a product of a reaction such as a detection sequence from an invasive cleavage reaction, a ligated probe from an OLA reaction, an extended probe from a PCR reaction, etc. Generally, as outlined herein, a target sequence from a sample is amplified to produce a secondary target that is detected, as outlined herein. Alternatively, an amplification step is done using a signal probe that is amplified, again producing a secondary target that is detected. The target sequence may be any length, with the understanding that longer sequences are more specific. As will be appreciated by those in the art, the complementary target sequence may take many forms. For example, it may be contained within a larger nucleic acid sequence, i.e. all or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, among others. As is outlined more fully below, probes are made to hybridize to target sequences to determine the presence or absence of the target sequence in a sample. Generally speaking, this term will be understood by those skilled in the art. The target sequence may also be comprised of different target domains; for example, in “sandwich” type assays as outlined below, a first target domain of the sample target sequence may hybridize to a capture probe or a portion of capture extender probe, a second target domain may hybridize to a portion of an amplifier probe, a label probe, or a different capture or capture extender probe, etc. In addition, the target domains may be adjacent (i.e. contiguous) or separated. For example, when LCR techniques are used, a first primer may hybridize to a first target domain and a second primer may hybridize to a second target domain; either the domains are adjacent, or they may be separated by one or more nucleotides, coupled with the use of a polymerase and dNTPs, as is more fully outlined below. The terms “first” and “second” are not meant to confer an orientation of the sequences with respect to the 5′-3′ orientation of the target sequence. For example, assuming a 5′-3′ orientation of the complementary target sequence, the first target domain may be located either 5′ to the second domain, or 3′ to the second domain. [0048]
  • If required, the target sequence is prepared using known techniques. For example, the sample may be treated to lyse the cells, using known lysis buffers, sonication, electroporation, etc., with purification occuring as needed, as will be appreciated by those in the art. In addition, the reactions outlined herein may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below. In addition, the reaction may include a variety of other reagents which may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc., which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target. [0049]
  • In addition, in most embodiments, double stranded target nucleic acids are denatured to render them single stranded so as to permit hybridization of the primers and other probes of the invention. A preferred embodiment utilizes a thermal step, generally by raising the temperature of the reaction to about 95° C., although pH changes and other techniques may also be used. [0050]
  • A primer nucleic acid is then contacted to the target sequence to form a hybridization complex. By “primer nucleic acid” herein is meant a probe nucleic acid that will hybridize to some portion, i.e. a domain, of the target sequence. Probes of the present invention are designed to be complementary to a target sequence (either the target sequence of the sample or to other probe sequences, as is described below), such that hybridization of the target sequence and the probes of the present invention occurs. As outlined below, this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence. Thus, by “substantially complementary” herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions. [0051]
  • A variety of hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al, hereby incorporated by reference. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g. 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g. greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of helix destabilizing agents such as formamide. The hybridization conditions may also vary when a non-ionic backbone, i.e. PNA is used, as is known in the art. In addition, cross-linking agents may be added after target binding to cross-link, i.e. covalently attach, the two strands of the hybridization complex. [0052]
  • Thus, the assays are generally run under stringency conditions which allows formation of the hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration, pH, organic solvent concentration, etc. [0053]
  • These parameters may also be used to control non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697. Thus it may be desirable to perform certain steps at higher stringency conditions to reduce non-specific binding. [0054]
  • The size of the primer nucleic acid may vary, as will be appreciated by those in the art, in general varying from 5 to 500 nucleotides in length, with primers of between 10 and 100 being preferred, between 15 and 50 being particularly preferred, and from 10 to 35 being especially preferred, depending on the use and amplification technique. [0055]
  • In addition, the different amplification techniques may have further requirements of the primers, as is more fully described below. [0056]
  • Once the hybridization complex between the primer and the target sequence has been formed, an enzyme, sometimes termed an “amplification enzyme”, is used to modify the primer. As for all the methods outlined herein, the enzymes may be added at any point during the assay, either prior to, during, or after the addition of the primers. The identity of the enzyme will depend on the amplification technique used, as is more fully outlined below. Similarly, the modification will depend on the amplification technique, as outlined below. [0057]
  • Once the enzyme has modified the primer to form a modified primer, the hybridization complex is disassociated. In one aspect, dissociation is by modification of the assay conditions. In another aspect, the modified primer no longer hybridizes to the target nucleic acid and dissociates. Either one or both of these aspects can be employed in signal and target amplification reactions as described below. Generally, the amplification steps are repeated for a period of time to allow a number of cycles, depending on the number of copies of the original target sequence and the sensitivity of detection, with cycles ranging from 1 to thousands, with from 10 to 100 cycles being preferred and from 20 to 50 cycles being especially preferred. [0058]
  • After a suitable time of amplification, unreacted primers are removed, in a variety of ways, as will be appreciated by those in the art and described below, and the hybridization complex is disassociated. In general, the modified primer comprises a detectable label, such as a fluorescent label, which is either incorporated by the enzyme or present on the original primer, and the modified primer is added to a microsphere array such is generally described in U.S. Ser. Nos. 09/189,543; 08/944,850; 09/033,462; 09/287,573; 09/151,877; 09/187,289 and 09/256,943; and PCT applications US98/09163 and US99/14387; US98/21193; US99/04473 and US98/05025, all of which are hereby incorporated by reference. The microsphere array comprises subpopulations of microspheres that comprise capture probes that will hybridize to the modified primers. Detection proceeds via detection of the label as an indication of the presence, absence or amount of the target sequence, as is more fully outlined below. [0059]
  • Target Amplification [0060]
  • In a preferred embodiment, the amplification is target amplification. Target amplification involves the amplification (replication) of the target sequence to be detected, such that the number of copies of the target sequence is increased. Suitable target amplification techniques include, but are not limited to, the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA) and nucleic acid sequence based amplification (NASBA). [0061]
  • Polymerase Chain Reaction Amplification [0062]
  • In a preferred embodiment, the target amplification technique is PCR. The polymerase chain reaction (PCR) is widely used and described, and involves the use of primer extension combined with thermal cycling to amplify a target sequence; see U.S. Pat. Nos. 4,683,195 and 4,683,202, and PCR Essential Data, J. W. Wiley & sons, Ed. C. R. Newton, 1995, all of which are incorporated by reference. In addition, there are a number of variations of PCR which also find use in the invention, including “quantitative competitive PCR” or “QC-PCR”, “arbitrarily primed PCR” or “AP-PCR”, “immuno-PCR”, “Alu-PCR”, “PCR single strand conformational polymorphism” or “PCR-SSCP”, “reverse transcriptase PCR” or “RT-PCR”, “biotin capture PCR”, “vectorette PCR”, “panhandle PCR”, and “PCR select cDNA subtraction”, “allele-specific PCR”, among others. [0063]
  • In general, PCR may be briefly described as follows. A double stranded target nucleic acid is denatured, generally by raising the temperature, and then cooled in the presence of an excess of a PCR primer, which then hybridizes to the first target strand. A DNA polymerase then acts to extend the primer with dNTPs, resulting in the synthesis of a new strand forming a hybridization complex. The sample is then heated again, to disassociate the hybridization complex, and the process is repeated. By using a second PCR primer for the complementary target strand, rapid and exponential amplification occurs. Thus PCR steps are denaturation, annealing and extension. The particulars of PCR are well known, and include the use of a thermostable polymerase such as Taq I polymerase and thermal cycling. [0064]
  • Accordingly, the PCR reaction requires at least one PCR primer, a polymerase, and a set of dNTPs. As outlined herein, the primers may comprise the label, or one or more of the dNTPs may comprise a label. [0065]
  • In general, as is more fully outlined below, the capture probes on the beads of the array are designed to be substantially complementary to the extended part of the primer; that is, unextended primers will not bind to the capture probes. Alternatively, as further described below, unreacted probes may be removed prior to addition to the array. [0066]
  • Strand Displacement Amplification (SDA) [0067]
  • In a preferred embodiment, the target amplification technique is SDA. Strand displacement amplification (SDA) is generally described in Walker et al., in Molecular Methods for Virus Detection, Academic Press, Inc., 1995, and U.S. Pat. Nos. 5,455,166 and 5,130,238, all of which are hereby expressly incorporated by reference in their entirety. [0068]
  • In general, SDA may be described as follows. A single stranded target nucleic acid, usually a DNA target sequence, is contacted with an SDA primer. An “SDA primer” generally has a length of 25-100 nucleotides, with SDA primers of approximately 35 nucleotides being preferred. An SDA primer is substantially complementary to a region at the 3′ end of the target sequence, and the primer has a sequence at its 5′ end (outside of the region that is complementary to the target) that is a recognition sequence for a restriction endonuclease, sometimes referred to herein as a “nicking enzyme” or a “nicking endonuclease”, as outlined below. The SDA primer then hybridizes to the target sequence. The SDA reaction mixture also contains a polymerase (an “SDA polymerase”, as outlined below) and a mixture of all four deoxynucleoside-triphosphates (also called deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP), at least one species of which is a substituted or modified dNTP; thus, the SDA primer is modified, i.e. extended, to form a modified primer, sometimes referred to herein as a “newly synthesized strand”. The substituted dNTP is modified such that it will inhibit cleavage in the strand containing the substituted dNTP but will not inhibit cleavage on the other strand. Examples of suitable substituted dNTPs include, but are not limited, 2′[0069] deoxyadenosine 5′-O-(1-thiotriphosphate), 5-methyideoxycytidine 5′-triphosphate, 2′-deoxyuridine 5′-triphosphate, adn 7-deaza-2′-deoxyguanosine 5′-triphosphate. In addition, the substitution of the dNTP may occur after incorporation into a newly synthesized strand; for example, a methylase may be used to add methyl groups to the synthesized strand. In addition, if all the nucleotides are substituted, the polymerase may have 5′→3′ exonuclease activity. However, if less than all the nucleotides are substituted, the polymerase preferably lacks 5′→3′ exonuclease activity.
  • As will be appreciated by those in the art, the recognition site/endonuclease pair can be any of a wide variety of known combinations. The endonuclease is chosen to cleave a strand either at the recognition site, or either 3′ or 5′ to it, without cleaving the complementary sequence, either because the enzyme only cleaves one strand or because of the incorporation of the substituted nucleotides. Suitable recognition site/endonuclease pairs are well known in the art; suitable endonucleases include, but are not limited to, HincII, HindII, AvaI, Fnu4HI, TthIIII, NcII, BstXI, BamHI, etc. A chart depicting suitable enzymes, and their corresponding recognition sites and the modified dNTP to use is found in U.S. Pat. No. 5,455,166, hereby expressly incorporated by reference. [0070]
  • Once nicked, a polymerase (an “SDA polymerase”) is used to extend the newly nicked strand, 5′→[0071] 3′, thereby creating another newly synthesized strand. The polymerase chosen should be able to intiate 5′→3′ polymerization at a nick site, should also displace the polymerized strand downstream from the nick, and should lack 5′→3′ exonuclease activity (this may be additionally accomplished by the addition of a blocking agent). Thus, suitable polymerases in SDA include, but are not limited to, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29 DNA polymerase.
  • Accordingly, the SDA reaction requires, in no particular order, an SDA primer, an SDA polymerase, a nicking endonuclease, and dNTPs, at least one species of which is modified. Again, as outlined above for PCR, preferred embodiments utilize capture probes complementary to the newly synthesized portion of the primer, rather than the primer region, to allow unextended primers to be removed. [0072]
  • In general, SDA does not require thermocycling. The temperature of the reaction is generally set to be high enough to prevent non-specific hybridization but low enough to allow specific hybridization; this is generally from about 37° C. to about 42° C., depending on the enzymes. [0073]
  • In a preferred embodiment, as for most of the amplification techniques described herein, a second amplification reaction can be done using the complementary target sequence, resulting in a substantial increase in amplification during a set period of time. That is, a second primer nucleic acid is hybridized to a second target sequence, that is substantially complementary to the first target sequence, to form a second hybridization complex. The addition of the enzyme, followed by disassociation of the second hybridization complex, results in the generation of a number of newly synthesized second strands. [0074]
  • Nucleic Acid Sequence Based Amplification (NASBA) and Transcription Mediated Amplification (TMA) [0075]
  • In a preferred embodiment, the target amplification technique is nucleic acid sequence based amplification (NASBA). NASBA is generally described in U.S. Pat. No. 5,409,818; Sooknanan et al., Nucleic Acid Sequence-Based Amplification, Ch. 12 (pp. 261-285) of Molecular Methods for Virus Detection, Academic Press, 1995; and “Profiting from Gene-based Diagnostics”, CTB International Publishing Inc., N.J., 1996, all of which are incorporated by reference. NASBA is very similar to both TMA and QBR. Transcription mediated amplification (TMA) is generally described in U.S. Pat. Nos. 5,399,491, 5,888,779, 5,705,365, 5,710,029, all of which are incorporated by reference. The main difference between NASBA and TMA is that NASBA utilizes the addition of RNAse H to effect RNA degradation, and TMA relies on inherent RNAse H activity of the reverse transcriptase. [0076]
  • In general, these techniques may be described as follows. A single stranded target nucleic acid, usually an RNA target sequence (sometimes referred to herein as “the first target sequence” or “the first template”), is contacted with a first primer, generally referred to herein as a “NASBA primer” (although “TMA primer” is also suitable). Starting with a DNA target sequence is described below. These primers generally have a length of 25-100 nucleotides, with NASBA primers of approximately 50-75 nucleotides being preferred. The first primer is preferably a DNA primer that has at its 3′ end a sequence that is substantially complementary to the 3′ end of the first template. The first primer also has an RNA polymerase promoter at its 5′ end (or its complement (antisense), depending on the configuration of the system). The first primer is then hybridized to the first template to form a first hybridization complex. The reaction mixture also includes a reverse transcriptase enzyme (an “NASBA reverse transcriptase”) and a mixture of the four dNTPs, such that the first NASBA primer is modified, i.e. extended, to form a modified first primer, comprising a hybridization complex of RNA (the first template) and DNA (the newly synthesized strand). [0077]
  • By “reverse transcriptase” or “RNA-directed DNA polymerase” herein is meant an enzyme capable of synthesizing DNA from a DNA primer and an RNA template. Suitable RNA-directed DNA polymerases include, but are not limited to, avian myloblastosis virus reverse transcriptase (“AMV RT”) and the Moloney murine leukemia virus RT. When the amplification reaction is TMA, the reverse transcriptase enzyme further comprises a RNA degrading activity as outlined below. [0078]
  • In addition to the components listed above, the NASBA reaction also includes an RNA degrading enzyme, also sometimes referred to herein as a ribonuclease, that will hydrolyze RNA of an RNA:DNA hybrid without hydrolyzing single- or double-stranded RNA or DNA. Suitable ribonucleases include, but are not limited to, RNase H from [0079] E. coli and calf thymus.
  • The ribonuclease activity degrades the first RNA template in the hybridization complex, resulting in a disassociation of the hybridization complex leaving a first single stranded newly synthesized DNA strand, sometimes referred to herein as “the second template”. [0080]
  • In addition, the NASBA reaction also includes a second NASBA primer, generally comprising DNA (although as for all the probes herein, including primers, nucleic acid analogs may also be used). This second NASBA primer has a sequence at its 3′ end that is substantially complementary to the 3′ end of the second template, and also contains an antisense sequence for a functional promoter and the antisense sequence of a transcription initiation site. Thus, this primer sequence, when used as a template for synthesis of the third DNA template, contains sufficient information to allow specific and efficient binding of an RNA polymerase and initiation of transcription at the desired site. Preferred embodiments utilizes the antisense promoter and transcription initiation site are that of the T7 RNA polymerase, although other RNA polymerase promoters and initiation sites can be used as well, as outlined below. [0081]
  • The second primer hybridizes to the second template, and a DNA polymerase, also termed a “DNA-directed DNA polymerase”, also present in the reaction, synthesizes a third template (a second newly synthesized DNA strand), resulting in second hybridization complex comprising two newly synthesized DNA strands. [0082]
  • Finally, the inclusion of an RNA polymerase and the required four ribonucleoside triphosphates (ribonucleotides or NTPs) results in the synthesis of an RNA strand (a third newly synthesized strand that is essentially the same as the first template). The RNA polymerase, sometimes referred to herein as a “DNA-directed RNA polymerase”, recognizes the promoter and specifically initiates RNA synthesis at the initiation site. In addition, the RNA polymerase preferably synthesizes several copies of RNA per DNA duplex. Preferred RNA polymerases include, but are not limited to, T7 RNA polymerase, and other bacteriophage RNA polymerases including those of phage T3, phage φII, Salmonella phage sp6, or Pseudomonase phage gh-1. [0083]
  • In some embodiments, TMA and NASBA are used with starting DNA target sequences. In this embodiment, it is necessary to utilize the first primer comprising the RNA polymerase promoter and a DNA polymerase enzyme to generate a double stranded DNA hybrid with the newly synthesized strand comprising the promoter sequence. The hybrid is then denatured and the second primer added. [0084]
  • Accordingly, the NASBA reaction requires, in no particular order, a first NASBA primer, a second NASBA primer comprising an antisense sequence of an RNA polymerase promoter, an RNA polymerase that recognizes the promoter, a reverse transcriptase, a DNA polymerase, an RNA degrading enzyme, NTPs and dNTPs, in addition to the detection components outlined below. [0085]
  • These components result in a single starting RNA template generating a single DNA duplex; however, since this DNA duplex results in the creation of multiple RNA strands, which can then be used to initiate the reaction again, amplification proceeds rapidly. [0086]
  • Accordingly, the TMA reaction requires, in no particular order, a first TMA primer, a second TMA primer comprising an antisense sequence of an RNA polymerase promoter, an RNA polymerase that recognizes the promoter, a reverse transcriptase with RNA degrading activity, a DNA polymerase, NTPs and dNTPs, in addition to the detection components outlined below. [0087]
  • These components result in a single starting RNA template generating a single DNA duplex; however, since this DNA duplex results in the creation of multiple RNA strands, which can then be used to initiate the reaction again, amplification proceeds rapidly. [0088]
  • As outlined herein, the detection of the newly synthesized strands can proceed in several ways. Direct detection can be done when the newly synthesized strands comprise detectable labels, either by incorporation into the primers or by incorporation of modified labelled nucleotides into the growing strand. Alternatively, as is more fully outlined below, indirect detection of unlabelled strands (which now serve as “targets” in the detection mode) can occur using a variety of sandwich assay configurations. As will be appreciated by those in the art, any of the newly synthesized strands can serve as the “target” for form an assay complex on a surface with a capture probe. In NASBA and TMA, it is preferable to utilize the newly formed RNA strands as the target, as this is where significant amplification occurs. [0089]
  • In this way, a number of secondary target molecules are made. As is more fully outlined below, these reactions (that is, the products of these reactions) can be detected in a number of ways. [0090]
  • Signal Amplification Techniques [0091]
  • In a preferred embodiment, the amplification technique is signal amplification. Signal amplification involves the use of limited number of target molecules as templates to either generate multiple signalling probes or allow the use of multiple signalling probes. Signal amplification strategies include LCR, CPT, QβR, invasive cleavage technology, and the use of amplification probes in sandwich assays. [0092]
  • Single Base Extension (SBE) [0093]
  • In a preferred embodiment, single base extension (SBE; sometimes referred to as “minisequencing”) is used for amplification. It should also be noted that SBE finds use in genotyping, as is described in co-pending application entitled “SEQUENCE DETERMINATION OF NUCLEIC ACIDS USING ARRAYS WITH MICROSPHERES” filed on Oct. 22, 1999 as U.S. Ser. No. 09/425,633. Briefly, SBE is a technique that utilizes an extension primer that hybridizes to the target nucleic acid. A polymerase (generally a DNA polymerase) is used to extend the 3′ end of the primer with a nucleotide analog labeled a detection label as described herein. Based on the fidelity of the enzyme, a nucleotide is only incorporated into the extension primer if it is complementary to the adjacent base in the target strand. Generally, the nucleotide is derivatized such that no further extensions can occur, so only a single nucleotide is added. However, for amplification reactions, this may not be necessary. Once the labeled nucleotide is added, detection of the label proceeds as outlined herein. See generally Sylvanen et al., Genomics 8:684-692 (1990); U.S. Pat. Nos. 5,846,710 and 5,888,819; Pastinen et al., Genomics Res. 7(6):606-614 (1997); all of which are expressly incorporated herein by reference. [0094]
  • The reaction is initiated by introducing the assay complex comprising the target sequence (i.e. the array) to a solution comprising a first nucleotide, frequently an nucleotide analog. By “nucleoide analog” in this context herein is meant a deoxynucleoside-triphosphate (also called deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP), that is further derivatized to be chain terminating. As will be appreciated by those in the art, any number of nucleotide analogs may be used, as long as a polymerase enzyme will still incorporate the nucleotide at the interrogation position. Preferred embodiments utilize dideoxy-triphosphate nucleotides (ddNTPs). Generally, a set of nucleotides comprising ddATP, ddCTP, ddGTP and ddTTP is used, at least one of which includes a label, and preferably all four. For amplification rather than genotyping reactions, the labels may all be the same; alternatively, different labels may be used. [0095]
  • In a preferred embodiment, the nucleotide analogs comprise a detectable label, which can be either a primary or secondary detectable label. Preferred primary labels are those outlined above. However, the enzymatic incorporation of nucleotides comprising fluorophores is poor under many conditions; accordingly, preferred embodiments utilize secondary detectable labels. In addition, as outlined below, the use of secondary labels may also facilitate the removal of unextended probes. [0096]
  • In addition to a first nucleotide, the solution also comprises an extension enzyme, generally a DNA polymerase. Suitable DNA polymerases include, but are not limited to, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical), T5 DNA polymerase and Phi29 DNA polymerase. If the NTP is complementary to the base of the detection position of the target sequence, which is adjacent to the extension primer, the extension enzyme will add it to the extension primer. Thus, the extension primer is modified, i.e. extended, to form a modified primer, sometimes referred to herein as a “newly synthesized strand”. [0097]
  • A limitation of this method is that unless the target nucleic acid is in sufficient concentration, the amount of unextended primer in the reaction greatly exceeds the resultant extended-labeled primer. The excess of unextended primer competes with the detection of the labeled primer in the assays described herein. Accordingly, when SBE is used, preferred embodiments utilize methods for the removal of unextended primers as outlined herein. [0098]
  • One method to overcome this limitation is thermocycling minisequencing in which repeated cycles of annealing, primer extension, and heat denaturation using a thermocycler and thermo-stable polymerase allows the amplification of the extension probe which results in the accumulation of extended primers. For example, if the original unextended primer to target nucleic acid concentration is 100:1 and 100 thermocycles and extensions are performed, a majority of the primer will be extended. [0099]
  • As will be appreciated by those in the art, the configuration of the SBE system can take on several forms. As for the LCR reaction described below, the reaction may be done in solution, and then the newly synthesized strands, with the base-specific detectable labels, can be detected. For example, they can be directly hybridized to capture probes that are complementary to the extension primers, and the presence of the label is then detected. [0100]
  • Alternatively, the SBE reaction can occur on a surface. For example, a target nucleic acid may be captured using a first capture probe that hybridizes to a first target domain of the target, and the reaction can proceed at a second target domain. The extended labeled primers are then bound to a second capture probe and detected. [0101]
  • Thus, the SBE reaction requires, in no particular order, an extension primer, a polymerase and dNTPs, at least one of which is labeled. [0102]
  • Oligonucleotide Ligation Amplification (OLA) [0103]
  • In a preferred embodiment, the signal amplification technique is OLA. OLA, which is referred to as the ligation chain reaction (LCR) when two-stranded substrates are used, involves the ligation of two smaller probes into a single long probe, using the target sequence as the template. In LCR, the ligated probe product becomes the predominant template as the reaction progresses. The method can be run in two different ways; in a first embodiment, only one strand of a target sequence is used as a template for ligation; alternatively, both strands may be used. See generally U.S. Pat. Nos. 5,185,243, 5,679,524 and 5,573,907; EP 0 320 308 B1; EP 0 336 731 B1; EP 0 439 182 B1; WO 90/01069; WO 89/12696; WO 97/31256; and WO 89/09835, and U.S. Ser. Nos. 60/078,102 and 60/073,011, all of which are incorporated by reference. [0104]
  • In a preferred embodiment, the single-stranded target sequence comprises a first target domain and a second target domain, which are adjacent and contiguous. A first OLA primer and a second OLA primer nucleic acids are added, that are substantially complementary to their respective target domain and thus will hybridize to the target domains. These target domains may be directly adjacent, i.e. contiguous, or separated by a number of nucleotides. If they are non-contiguous, nucleotides are added along with means to join nucleotides, such as a polymerase, that will add the nucleotides to one of the primers. The two OLA primers are then covalently attached, for example using a ligase enzyme such as is known in the art, to form a modified primer. This forms a first hybridization complex comprising the ligated probe and the target sequence. This hybridization complex is then denatured (disassociated), and the process is repeated to generate a pool of ligated probes. [0105]
  • In a preferred embodiment, OLA is done for two strands of a double-stranded target sequence. The target sequence is denatured, and two sets of probes are added: one set as outlined above for one strand of the target, and a separate set (i.e. third and fourth primer probe nucleic acids) for the other strand of the target. In a preferred embodiment, the first and third probes will hybridize, and the second and fourth probes will hybridize, such that amplification can occur. That is, when the first and second probes have been attached, the ligated probe can now be used as a template, in addition to the second target sequence, for the attachment of the third and fourth probes. Similarly, the ligated third and fourth probes will serve as a template for the attachment of the first and second probes, in addition to the first target strand. In this way, an exponential, rather than just a linear, amplification can occur. [0106]
  • As will be appreciated by those in the art, the ligation product can be detected in a variety of ways. In a preferred embodiment, the ligation reaction is run in solution. In this embodiment, only one of the primers carries a detectable label, e.g. the first ligation probe, and the capture probe on the bead is substantially complementary to the other probe, e.g. the second ligation probe. In this way, unextended labeled ligation primers will not interfere with the assay. That is, in a preferred embodiment, the ligation product is detected by solid-phase oligonucleotide probes. The solid-phase probes are preferably complementary to at least a portion of the ligation product. In a preferred embodiment, the solid-phase probe is complementary to the 5′ detection oligonucleotide portion of the ligation product. This substantially reduces or eliminates false signal generated by the optically-labeled 3′ primers. Preferably, detection is accomplished by removing the unligated 5′ detection oligonucleotide from the reaction before application to a capture probe. In one embodiment, the unligated 5′ detection oligonucleotides are removed by digesting 3′ non-protected oligonucleotides with a 3′ exonuclease, such as, exonuclease I. The ligation products are protected from exo I digestion by including, for example, 4-phosphorothioate residues at their 3′ terminus, thereby, rendering them resistant to exonuclease digestion. The unligated detection oligonucleotides are not protected and are digested. [0107]
  • Alternatively, the target nucleic acid is immobilized on a solid-phase surface. The ligation assay is performed and unligated oligonucleotides are removed by washing under appropriate stringency to remove unligated oligonucleotides. The ligated oligonucleotides are eluted from the target nucleic acid using denaturing conditions, such as, 0.1 N NaOH, and detected as described herein. [0108]
  • Again, as outlined above, the detection of the LCR reaction can also occur directly, in the case where one or both of the primers comprises at least one detectable label, or indirectly, using sandwich assays, through the use of additional probes; that is, the ligated probes can serve as target sequences, and detection may utilize amplification probes, capture probes, capture extender probes, label probes, and label extender probes, etc. [0109]
  • Rolling-Circle Amplification (RCA) [0110]
  • In a preferred embodiment the signal amplification technique is RCA. Rolling-circle amplification is generally described in Baner et al. (1998) [0111] Nuc. Acids Res. 26:5073-5078; Barany, F. (1991) Proc. Natl. Acad. Sci. USA 88:189-193; and Lizardi et al. (1998) Nat. Genet. 19:225-232, all of which are incorporated by reference in their entirety.
  • In general, RCA may be described in two ways. First, as is outlined in more detail below, a single probe is hybridized with a target nucleic acid. Each terminus of the probe hybridizes adjacently on the target nucleic acid and the OLA assay as described above occurs. When ligated, the probe is circularized while hybridized to the target nucleic acid. Addition of a polymerase results in extension of the circular probe. However, since the probe has no terminus, the polymerase continues to extend the probe repeatedly. Thus results in amplification of the circular probe. [0112]
  • A second alternative approach involves OLA followed by RCA. In this embodiment, an immobilized primer is contacted with a target nucleic acid. Complementary sequences will hybridize with each other resulting in an immobilized duplex. A second primer is contacted with the target nucleic acid. The second primer hybridizes to the target nucleic acid adjacent to the first primer. An OLA assay is performed as described above. Ligation only occurs if the primer are complementary to the target nucleic acid. When a mismatch occurs, particularly at one of the nucleotides to be ligated, ligation will not occur. Following ligation of the oligonucleotides, the ligated, immobilized, oligonucleotide is then hybridized with an RCA probe. This is a circular probe that is designed to specifically hybridize with the ligated oligonucleotide and will only hybridize with an oligonucleotide that has undergone ligation. RCA is then performed as is outlined in more detail below. [0113]
  • Accordingly, in an preferred embodiment, a single oligonucleotide is used both for OLA and as the circular template for RCA (referred to herein as a “padlock probe” or a “RCA probe”). That is, each terminus of the oligonucleotide contains sequence complementary to the target nucleic acid and functions as an OLA primer as described above. That is, the first end of the RCA probe is substantially complementary to a first target domain, and the second end of the RCA probe is substantially complementary to a second target domain, adjacent to the first domain. Hybridization of the oligonucleotide to the target nucleic acid results in the formation of a hybridization complex. Ligation of the “primers” (which are the discrete ends of a single oligonucleotide) results in the formation of a modified hybridization complex containing a circular probe i.e. an RCA template complex. That is, the oligonucleotide is circularized while still hybridized with the target nucleic acid. This serves as a circular template for RCA. Addition of a polymerase to the RCA template complex results in the formation of an amplified product nucleic acid. Following RCA, the amplified product nucleic acid is detected (FIG. 6). This can be accomplished in a variety of ways; for example, the polymerase may incorporate labelled nucleotides, or alternatively, a label probe is used that is substantially complementary to a portion of the RCA probe and comprises at least one label is used. [0114]
  • The polymerase can be any polymerase, but is preferably one lacking 3′ exonuclease activity (3′ exo[0115] ). Examples of suitable polymerase include but are not limited to exonuclease minus DNA Polymerase I large (Klenow) Fragment, Phi29 DNA polymerase, Taq DNA Polymerase and the like. In addition, in some embodiments, a polymerase that will replicate single-stranded DNA (i.e. without a primer forming a double stranded section) can be used.
  • In a preferred embodiment, the RCA probe contains an adapter sequence as outlined herein, with adapter capture probes on the array, for example on a microsphere when microsphere arrays are being used. Alternatively, unique portions of the RCA probes, for example all or part of the sequence corresponding to the target sequence, can be used to bind to a capture probe. [0116]
  • In a preferred embodiment, the padlock probe contains a restriction site. The restriction endonuclease site allows for cleavage of the long concatamers that are typically the result of RCA into smaller individual units that hybridize either more efficiently or faster to surface bound capture probes. Thus, following RCA, the product nucleic acid is contacted with the appropriate restriction endonuclease. This results in cleavage of the product nucleic acid into smaller fragments. The fragments are then hybridized with the capture probe that is immobilized resulting in a concentration of product fragments onto the microsphere. Again, as outlined herein, these fragments can be detected in one of two ways: either labelled nucleotides are incorporated during the replication step, or an additional label probe is added. [0117]
  • Thus, in a preferred embodiment, the padlock probe comprises a label sequence; i.e. a sequence that can be used to bind label probes and is substantially complementary to a label probe. In one embodiment, it is possible to use the same label sequence and label probe for all padlock probes on an array; alternatively, each padlock probe can have a different label sequence. [0118]
  • The padlock probe also contains a priming site for priming the RCA reaction. That is, each padlock probe comprises a sequence to which a primer nucleic acid hybridizes forming a template for the polymerase. The primer can be found in any portion of the circular probe. In a preferred embodiment, the primer is located at a discrete site in the probe. In this embodiment, the primer site in each distinct padlock probe is identical, although this is not required. Advantages of using primer sites with identical sequences include the ability to use only a single primer oligonucleotide to prime the RCA assay with a plurality of different hybridization complexes. That is, the padlock probe hybridizes uniquely to the target nucleic acid to which it is designed. A single primer hybridizes to all of the unique hybridization complexes forming a priming site for the polymerase. RCA then proceeds from an identical locus within each unique padlock probe of the hybridization complexes. [0119]
  • In an alternative embodiment, the primer site can overlap, encompass, or reside within any of the above-described elements of the padlock probe. That is, the primer can be found, for example, overlapping or within the restriction site or the identifier sequence. In this embodiment, it is necessary that the primer nucleic acid is designed to base pair with the chosen primer site. [0120]
  • Thus, the padlock probe of the invention contains at each terminus, sequences corresponding to OLA primers. The intervening sequence of the padlock probe contain in no particular order, an adapter sequence and a restriction endonuclease site. In addition, the padlock probe contains a RCA priming site. [0121]
  • Thus, in a preferred embodiment the OLA/RCA is performed in solution followed by restriction endonuclease cleavage of the RCA product. The cleaved product is then applied to an array comprising beads, each bead comprising a probe complementary to the adapter sequence located in the padlock probe. The amplified adapter sequence correlates with a particular target nucleic acid. Thus the incorporation of an endonuclease site allows the generation of short, easily hybridizable sequences. Furthermore, the unique adapter sequence in each rolling circle padlock probe sequence allows diverse sets of nucleic acid sequences to be analyzed in parallel on an array, since each sequence is resolved on the basis of hybridization specificity. [0122]
  • In an alternative OLA/RCA method, one of the OLA primers is immobilized on the microsphere; the second primer is added in solution. Both primers hybridize with the target nucleic acid forming a hybridization complex as described above for the OLA assay. [0123]
  • As described herein, the microsphere is distributed on an array. In a preferred embodiment, a plurality of microspheres each with a unique OLA primer is distributed on the array. [0124]
  • Following the OLA assay, and either before, after or concurrently with distribution of the beads on the array, a segment of circular DNA is hybridized to the bead-based ligated oligonucleotide forming a modified hybridization complex. Addition of an appropriate polymerase (3′ exo[0125] ), as is known in the art, and corresponding reaction buffer to the array leads to amplification of the circular DNA. Since there is no terminus to the circular DNA, the polymerase continues to travel around the circular template generating extension product until it detaches from the template. Thus, a polymerase with high processivity can create several hundred or thousand copies of the circular template with all the copies linked in one contiguous strand.
  • Again, these copies are subsequently detected by one of two methods; either hybridizing a labeled oligo complementary to the circular target or via the incorporation of labeled nucleotides in the amplification reaction. The label is detected using conventional label detection methods as described herein. [0126]
  • In one embodiment, when the circular DNA contains sequences complementary to the ligated oligonucleotide it is preferable to remove the target DNA prior to contacting the ligated oligonucleotide with the circular DNA (See FIG. 7). This is done by denaturing the double-stranded DNA by methods known in the art. In an alternative embodiment, the double stranded DNA is not denatured prior to contacting the circular DNA. [0127]
  • In an alternative embodiment, when the circular DNA contains sequences complementary to the target nucleic acid, it is preferable that the circular DNA is complementary at a site distinct from the site bound to the ligated oligonucleotide. In this embodiment it is preferred that the duplex between the ligated oligonucleotide and target nucleic acid is not denatured or disrupted prior to the addition of the circular DNA so that the target DNA remains immobilized to the bead. [0128]
  • Hybridization and washing conditions are well known in the art; various degrees of stringency can be used. In some embodiments it is not necessary to use stringent hybridization or washing conditions as only microspheres containing the ligated probes will effectively hybridize with the circular DNA; microspheres bound to DNA that did not undergo ligation (those without the appropriate target nucleic acid) will not hybridize as strongly with the circular DNA as those primers that were ligated. Thus, hybridization and/or washing conditions are used that discriminate between binding of the circular DNA to the ligated primer and the unligated primer. [0129]
  • Alternatively, when the circular probe is designed to hybridize to the target nucleic acid at a site distinct from the site bound to the ligated oligonucleotide, hybridization and washing conditions are used to remove or dissociate the target nucleic acid from unligated oligonucleotides while target nucleic acid hybridizing with the ligated oligonucleotides will remain bound to the beads. In this embodiment, the circular probe only hybridizes to the target nucleic acid when the target nucleic acid is hybridized with a ligated oligonucleotide that is immobilized on a bead. [0130]
  • As is well known in the art, an appropriate polymerase (3′ exo[0131] ) is added to the array. The polymerase extends the sequence of a single-stranded DNA using double-stranded DNA as a primer site. In one embodiment, the circular DNA that has hybridized with the appropriate OLA reaction product serves as the primer for the polymerase. In the presence of an appropriate reaction buffer as is known in the art, the polymerase will extend the sequence of the primer using the single-stranded circular DNA as a template. As there is no terminus of the circular DNA, the polymerase will continue to extend the sequence of the circular DNA. In an alternative embodiment, the RCA probe comprises a discrete primer site located within the circular probe. Hybridization of primer nucleic acids to this primer site forms the polymerase template allowing RCA to proceed.
  • In a preferred embodiment, the polymerase creates more than 100 copies of the circular DNA. In more preferred embodiments the polymerase creates more than 1000 copies of the circular DNA; while in a most preferred embodiment the polymerase creates more than 10,000 copies or more than 50,000 copies of the template. [0132]
  • The amplified circular DNA sequence is then detected by methods known in the art and as described herein. Detection is accomplished by hybridizing with a labeled probe. The probe is labeled directly or indirectly. Alternatively, labeled nucleotides are incorporated into the amplified circular DNA product. The nucleotides can be labeled directly, or indirectly as is further described herein. [0133]
  • The RCA as described herein finds use in allowing highly specific and highly sensitive detection of nucleic acid target sequences. In particular, the method finds use in improving the multiplexing ability of DNA arrays and eliminating costly sample or target preparation. As an example, a substantial savings in cost can be realized by directly analyzing genomic DNA on an array, rather than employing an intermediate PCR amplification step. The method finds use in examining genomic DNA and other samples including mRNA. [0134]
  • In addition the RCA finds use in allowing rolling circle amplification products to be easily detected by hybridization to probes in a solid-phase format (e.g. an array of beads). An additional advantage of the RCA is that it provides the capability of multiplex analysis so that large numbers of sequences can be analyzed in parallel. By combining the sensitivity of RCA and parallel detection on arrays, many sequences can be analyzed directly from genomic DNA. [0135]
  • Chemical Ligation Techniques [0136]
  • A variation of LCR utilizes a “chemical ligation” of sorts, as is generally outlined in U.S. Pat. Nos. 5,616,464 and 5,767,259, both of which are hereby expressly incorporated by reference in their entirety. In this embodiment, similar to enzymatic ligation, a pair of primers are utilized, wherein the first primer is substantially complementary to a first domain of the target and the second primer is substantially complementary to an adjacent second domain of the target (although, as for enzymatic ligation, if a “gap” exists, a polymerase and dNTPs may be added to “fill in” the gap). Each primer has a portion that acts as a “side chain” that does not bind the target sequence and acts as one half of a stem structure that interacts non-covalently through hydrogen bonding, salt bridges, van der Waal's forces, etc. Preferred embodiments utilize substantially complementary nucleic acids as the side chains. Thus, upon hybridization of the primers to the target sequence, the side chains of the primers are brought into spatial proximity, and, if the side chains comprise nucleic acids as well, can also form side chain hybridization complexes. [0137]
  • At least one of the side chains of the primers comprises an activatable cross-linking agent, generally covalently attached to the side chain, that upon activation, results in a chemical cross-link or chemical ligation. The activatible group may comprise any moiety that will allow cross-linking of the side chains, and include groups activated chemically, photonically and thermally, with photoactivatable groups being preferred. In some embodiments a single activatable group on one of the side chains is enough to result in cross-linking via interaction to a functional group on the other side chain; in alternate embodiments, activatable groups are required on each side chain. [0138]
  • Once the hybridization complex is formed, and the cross-linking agent has been activated such that the primers have been covalently attached, the reaction is subjected to conditions to allow for the disassocation of the hybridization complex, thus freeing up the target to serve as a template for the next ligation or cross-linking. In this way, signal amplification occurs, and can be detected as outlined herein. [0139]
  • Invasive Cleavage Techniques [0140]
  • In a preferred embodiment, the signal amplification technique is invasive cleavage technology, which is described in a number of patents and patent applications, including U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669, all of which are hereby incorporated by reference in their entirety. [0141]
  • Generally, invasive cleavage technology may be described as follows. A target nucleic acid is recognized by two distinct probes. A first probe, generally referred to herein as an “invader” probe, is substantially complementary to a first portion of the target nucleic acid. A second probe, generally referred to herein as a “signal probe”, is partially complementary to the target nucleic acid; the 3′ end of the signal oligonucleotide is substantially complementary to the target sequence while the 5′ end is non-complementary and preferably forms a single-stranded “tail” or “arm”. The non-complementary end of the second probe preferably comprises a “generic” or “unique” sequence, frequently referred to herein as a “detection sequence”, that is used to indicate the presence or absence of the target nucleic acid, as described below. The detection sequence of the second probe preferably comprises at least one detectable label, although as outlined herein, since this detection sequence can function as a target sequence for a capture probe, sandwich configurations utilizing label probes as described herein may also be done. [0142]
  • Hybridization of the first and second oligonucleotides near or adjacent to one another on the target nucleic acid forms a number of structures. In a preferred embodiment, a forked cleavage structure forms and is a substrate of a nuclease which cleaves the detection sequence from the signal oligonucleotide. The site of cleavage is controlled by the distance or overlap between the 3′ end of the invader oligonucleotide and the downstream fork of the signal oligonucleotide. Therefore, neither oligonucleotide is subject to cleavage when misaligned or when unattached to target nucleic acid. [0143]
  • In a preferred embodiment, the nuclease that recognizes the forked cleavage structure and catalyzes release of the tail is thermostable, thereby, allowing thermal cycling of the cleavage reaction, if desired. Preferred nucleases derived from thermostable DNA polymerases that have been modified to have reduced synthetic activity which is an undesirable side-reaction during cleavage are disclosed in U.S. Pat. Nos. 5,719,028 and 5,843,669, hereby expressly by reference. The synthetic activity of the DNA polymerase is reduced to a level where it does not interfere with detection of the cleavage reaction and detection of the freed tail. Preferably the DNA polymerase has no detectable polymerase activity. Examples of nucleases are those derived from [0144] Thermus aquaticus, Thermus flavus, or Thermus thermophilus.
  • In another embodiment, thermostable structure-specific nucleases are Flap endonucleases (FENs) selected from FEN-1 or FEN-2 like (e.g. XPG and RAD2 nucleases) from Archaebacterial species, for example, FEN-1 from [0145] Methanococcus jannaschii, Pyrococcus furiosis, Pyrococcus woesei, and Archaeoglobus fulgidus. (U.S. Pat. No. 5,843,669 and Lyamichev et al. 1999. Nature Biotechnology 17:292-297; both of which are hereby expressly by reference).
  • In a preferred embodiment, the nuclease is AfuFEN1 or PfuFEN1 nuclease. To cleave a forked structure, these nucleases require at least one overlapping nucleotide between the signal and invasive probes to recognize and cleave the 5′ end of the signal probe. To effect cleavage the 3′-terminal nucleotide of the invader oligonucleotide is not required to be complementary to the target nucleic acid. In contast, mismatch of the signal probe one base upstream of the cleavage site prevents creation of the overlap and cleavage. The specificity of the nuclease reaction allows single nucleotide polymorphism (SNP) detection from, for example, genomic DNA, as outlined below (Lyamichev et al.). [0146]
  • In a preferred embodiment invasive cleavage technology is used. Invasive cleavage technology is based on structure-specific nucleases that cleave nucleic acids in a site-specific manner. Two probes are used: an “invader” probe and a “signalling” probe, that adjacently hybridize to a target sequence with overlap. For mismatch discrimination, the invader technology relies on complementarity at the overlap position where cleavage occurs. The enzyme cleaves at the overlap, and releases the “tail” which may or may not be labeled. This can then be detected. The Invader™ technology is described in U.S. Pat. Nos. 5,846,717; 5,614,402; 5,719,028; 5,541,311; and 5,843,669, all of which are hereby incorporated by reference. [0147]
  • The invasive cleavage assay is preferably performed on an array format. In a preferred embodiment, the signal probe has a detectable label, attached 5′ from the site of nuclease cleavage (e.g. within the detection sequence) and a capture tag, as described below (e.g. biotin or other hapten) 3′ from the site of nuclease cleavage. After the assay is carried out, the 3′ portion of the cleaved signal probe (e.g. the the detection sequence) are extracted, for example, by binding to streptavidin beads or by crosslinking through the capture tag to produce aggregates or by antibody to an attached hapten. By “capture tag” herein is a meant one of a pair of binding partners as described above, such as antigen/antibody pairs, digoxygenenin, dinitrophenol, etc. [0148]
  • The cleaved 5′ region, e.g. the detection sequence, of the signal probe, comprises a label and is detected and optionally quantitated. In one embodiment, the cleaved 5′ region is hybridized to a probe on an array (capture probe) and optically detected. As described below, many signal probes can be analyzed in parallel by hybridization to their complementary probes in an array. [0149]
  • In a preferred embodiment, the invasive cleavage reaction is configured to utilize a fluorophore-quencher reaction. A signalling probe comprising both a fluorophore and a quencher is used, with the fluorophore and the quencher on opposite sides of the cleavage site. As will be appreciated by those in the art, these will be positioned closely together. Thus, in the absence of cleavage, very little signal is seen due to the quenching reaction. After cleavage, however, the distance between the two is large, and thus fluorescence can be detected. Upon assembly of an assay complex, comprising the target sequence, an invader probe, and a signalling probe, and the introduction of the cleavage enzyme, the cleavage of the complex results in the disassociation of the quencher from the complex, resulting in an increase in fluorescence. [0150]
  • In this embodiment, suitable fluorophore-quencher pairs are as known in the art. For example, suitable quencher molecules comprise Dabcyl. [0151]
  • As will be appreciated by those in the art, this system can be configured in a variety of conformations, as discussed in FIG. 4. [0152]
  • In a preferred embodiment, to obtain higher specificity and reduce the detection of contaminating uncleaved signal probe or incorrectly cleaved product, an additional enzymatic recognition step is introduced in the array capture procedure. For example, the cleaved signal probe binds to a capture probe to produce a double-stranded nucleic acid in the array. In this embodiment, the 3′ end of the cleaved signal probe is adjacent to the 5′ end of one strand of the capture probe, thereby, forming a substrate for DNA ligase (Broude et al. 1991. PNAS 91: 3072-3076). Only correctly cleaved product is ligated to the capture probe. Other incorrectly hybridized and non-cleaved signal probes are removed, for example, by heat denaturation, high stringency washes, and other methods that disrupt base pairing. [0153]
  • Cycling Probe Techniques (CPT) [0154]
  • In a preferred embodiment, the signal amplification technique is CPT. CPT technology is described in a number of patents and patent applications, including U.S. Pat. Nos. 5,011,769, 5,403,711, 5,660,988, and 4,876,187, and PCT published applications WO 95/05480, WO 95/1416, and WO 95/00667, and U.S. Ser. No. 09/014,304, all of which are expressly incorporated by reference in their entirety. [0155]
  • Generally, CPT may be described as follows. A CPT primer (also sometimes referred to herein as a “scissile primer”), comprises two probe sequences separated by a scissile linkage. The CPT primer is substantially complementary to the target sequence and thus will hybridize to it to form a hybridization complex. The scissile linkage is cleaved, without cleaving the target sequence, resulting in the two probe sequences being separated. The two probe sequences can thus be more easily disassociated from the target, and the reaction can be repeated any number of times. The cleaved primer is then detected as outlined herein. [0156]
  • By “scissile linkage” herein is meant a linkage within the scissile probe that can be cleaved when the probe is part of a hybridization complex, that is, when a double-stranded complex is formed. It is important that the scissile linkage cleave only the scissile probe and not the sequence to which it is hybridized (i.e. either the target sequence or a probe sequence), such that the target sequence may be reused in the reaction for amplification of the signal. As used herein, the scissile linkage, is any connecting chemical structure which joins two probe sequences and which is capable of being selectively cleaved without cleavage of either the probe sequences or the sequence to which the scissile probe is hybridized. The scissile linkage may be a single bond, or a multiple unit sequence. As will be appreciated by those in the art, a number of possible scissile linkages may be used. [0157]
  • In a preferred embodiment, the scissile linkage comprises RNA. This system, previously described in as outlined above, is based on the fact that certain double-stranded nucleases, particularly ribonucleases, will nick or excise RNA nucleosides from a RNA:DNA hybridization complex. Of particular use in this embodiment is RNAseH, Exo III, and reverse transcriptase. [0158]
  • In one embodiment, the entire scissile probe is made of RNA, the nicking is facilitated especially when carried out with a double-stranded ribonuclease, such as RNAseH or Exo III. RNA probes made entirely of RNA sequences are particularly useful because first, they can be more easily produced enzymatically, and second, they have more cleavage sites which are accessible to nicking or cleaving by a nicking agent, such as the ribonucleases. Thus, scissile probes made entirely of RNA do not rely on a scissile linkage since the scissile linkage is inherent in the probe. [0159]
  • In a preferred embodiment, when the scissile linkage is a nucleic acid such as RNA, the methods of the invention may be used to detect mismatches, as is generally described in U.S. Pat. Nos. 5,660,988, and WO 95/14106, hereby expressly incorporated by reference. These mismatch detection methods are based on the fact that RNAseH may not bind to and/or cleave an RNA:DNA duplex if there are mismatches present in the sequence. Thus, in the NA[0160] 1-R-NA2 embodiments, NA1 and NA2 are non-RNA nucleic acids, preferably DNA. Preferably, the mismatch is within the RNA:DNA duplex, but in some embodiments the mismatch is present in an adjacent sequence very close to the desired sequence, close enough to affect the RNAseH (generally within one or two bases). Thus, in this embodiment, the nucleic acid scissile linkage is designed such that the sequence of the scissile linkage reflects the particular sequence to be detected, i.e. the area of the putative mismatch.
  • In some embodiments of mismatch detection, the rate of generation of the released fragments is such that the methods provide, essentially, a yes/no result, whereby the detection of virtually any released fragment indicates the presence of the desired target sequence. Typically, however, when there is only a minimal mismatch (for example, a 1-, 2- or 3-base mismatch, or a 3-base deletion), there is some generation of cleaved sequences even though the target sequence is not present. Thus, the rate of generation of cleaved fragments, and/or the final amount of cleaved fragments, is quantified to indicate the presence or absence of the target. In addition, the use of secondary and tertiary scissile probes may be particularly useful in this embodiment, as this can amplify the differences between a perfect match and a mismatch. These methods may be particularly useful in the determination of homozygotic or heterozygotic states of a patient. [0161]
  • In this embodiment, it is an important feature of the scissile linkage that its length is determined by the suspected difference between the target and the probe. In particular, this means that the scissile linkage must be of sufficient length to encompass the suspected difference, yet short enough so that the scissile linkage cannot inappropriately “specifically hybridize” to the selected nucleic acid molecule when the suspected difference is present; such inappropriate hybridization would permit excision and thus cleavage of scissile linkages even though the selected nucleic acid molecule was not fully complementary to the nucleic acid probe. Thus in a preferred embodiment, the scissile linkage is between 3 to 5 nucleotides in length, such that a suspected nucleotide difference from 1 nucleotide to 3 nucleotides is encompassed by the scissile linkage, and 0, 1 or 2 nucleotides are on either side of the difference. [0162]
  • Thus, when the scissile linkage is nucleic acid, preferred embodiments utilize from 1 to about 100 nucleotides, with from about 2 to about 20 being preferred and from about 5 to about 10 being particularly preferred. [0163]
  • CPT may be done enzymatically or chemically. That is, in addition to RNAseH, there are several other cleaving agents which may be useful in cleaving RNA (or other nucleic acid) scissile bonds. For example, several chemical nucleases have been reported; see for example Sigman et al., Annu. Rev. Biochem. 1990, 59, 207-236; Sigman et al., Chem. Rev. 1993, 93, 2295-2316; Bashkin et al., J. Org. Chem. 1990, 55, 5125-5132; and Sigman et al., Nucleic Acids and Molecular Biology, vol. 3, F. Eckstein and D. M. J. Lilley (Eds), Springer-Verlag, Heidelberg 1989, pp. 13-27; all of which are hereby expressly incorporated by reference. [0164]
  • Specific RNA hydrolysis is also an active area; see for example Chin, Acc. Chem. Res. 1991, 24, 145-152; Breslow et al., Tetrahedron, 1991, 47, 2365-2376; Anslyn et al., Angew. Chem. Int. Ed. Engl., 1997, 36, 432-450; and references therein, all of which are expressly incorporated by reference. Reactive phosphate centers are also of interest in developing scissile linkages, see Hendry et al., Prog. Inorg. Chem.: Bioinorganic Chem. 1990, 31, 201-258 also expressly incorporated by reference. [0165]
  • Current approaches to site-directed RNA hydrolysis include the conjugation of a reactive moiety capable of cleaving phosphodiester bonds to a recognition element capable of sequence-specifically hybridizing to RNA. In most cases, a metal complex is covalently attached to a DNA strand which forms a stable heteroduplex. Upon hybridization, a Lewis acid is placed in close proximity to the RNA backbone to effect hydrolysis; see Magda et al., J. Am. Chem. Soc. 1994, 116, 7439; Hall et al., Chem. Biology 1994, 1, 185-190; Bashkin et al., J. Am. Chem. Soc. 1994, 116, 5981-5982; Hall et al., Nucleic Acids Res. 1996, 24, 3522; Magda et al., J. Am. Chem. Soc. 1997, 119, 2293; and Magda et al., J. Am. Chem. Soc. 1997, 119, 6947, all of which are expressly incorporated by reference. [0166]
  • In a similar fashion, DNA-polyamine conjugates have been demonstrated to induce site-directed RNA strand scission; see for example, Yoshinari et al., J. Am. Chem. Soc. 1991, 113, 5899-5901; Endo et al., J. Org. Chem. 1997, 62, 846; and Barbier et al., J. Am. Chem. Soc. 1992, 114, 3511-3515, all of which are expressly incorporated by reference. [0167]
  • In a preferred embodiment, the scissile linkage is not necessarily RNA. For example, chemical cleavage moieties may be used to cleave basic sites in nucleic acids; see Belmont, et al.,New J. Chem. 1997, 21, 47-54; and references therein, all of which are expressly incorporated herein by reference. Similarly, photocleavable moieties, for example, using transition metals, may be used; see Moucheron, et al., Inorg. Chem. 1997, 36, 584-592, hereby expressly by reference. [0168]
  • Other approaches rely on chemical moieties or enzymes; see for example Keck et al., Biochemistry 1995, 34, 12029-12037; Kirk et al., Chem. Commun. 1998, in press; cleavage of G-U basepairs by metal complexes; see Biochemistry, 1992, 31, 5423-5429; diamine complexes for cleavage of RNA; Komiyama, et al., J. Org. Chem. 1997, 62, 2155-2160; and Chow et al., Chem. Rev. 1997, 97, 1489-1513, and references therein, all of which are expressly incorporated herein by reference. [0169]
  • The first step of the CPT method requires hybridizing a primary scissile primer (also called a primary scissile probe) to the target. This is preferably done at a temperature that allows both the binding of the longer primary probe and disassociation of the shorter cleaved portions of the primary probe, as will be appreciated by those in the art. As outlined herein, this may be done in solution, or either the target or one or more of the scissile probes may be attached to a solid support. For example, it is possible to utilize “anchor probes” on a solid support which are substantially complementary to a portion of the target sequence, preferably a sequence that is not the same sequence to which a scissile probe will bind. [0170]
  • Similarly, as outlined herein, a preferred embodiment has one or more of the scissile probes attached to a solid support such as a bead. In this embodiment, the soluble target diffuses to allow the formation of the hybridization complex between the soluble target sequence and the support-bound scissile probe. In this embodiment, it may be desirable to include additional scissile linkages in the scissile probes to allow the release of two or more probe sequences, such that more than one probe sequence per scissile probe may be detected, as is outlined below, in the interests of maximizing the signal. [0171]
  • In this embodiment (and in other amplification techniques herein), preferred methods utilize cutting or shearing techniques to cut the nucleic acid sample containing the target sequence into a size that will allow sufficient diffusion of the target sequence to the surface of a bead. This may be accomplished by shearing the nucleic acid through mechanical forces (e.g. sonication) or by cleaving the nucleic acid using restriction endonucleases. Alternatively, a fragment containing the target may be generated using polymerase, primers and the sample as a template, as in polymerase chain reaction (PCR). In addition, amplification of the target using PCR or LCR or related methods may also be done; this may be particularly useful when the target sequence is present in the sample at extremely low copy numbers. Similarly, numerous techniques are known in the art to increase the rate of mixing and hybridization including agitation, heating, techniques that increase the overall concentration such as precipitation, drying, dialysis, centrifugation, electrophoresis, magnetic bead concentration, etc. [0172]
  • In general, the scissile probes are introduced in a molar excess to their targets (including both the target sequence or other scissile probes, for example when secondary or tertiary scissile probes are used), with ratios of scissile probe:target of at least about 100:1 being preferred, at least about 1000:1 being particularly preferred, and at least about 10,000:1 being especially preferred. In some embodiments the excess of probe:target will be much greater. In addition, ratios such as these may be used for all the amplification techniques outlined herein. [0173]
  • Once the hybridization complex between the primary scissile probe and the target has been formed, the complex is subjected to cleavage conditions. As will be appreciated, this depends on the composition of the scissile probe; if it is RNA, RNAseH is introduced. It should be noted that under certain circumstances, such as is generally outlined in WO 95/00666 and WO 95/00667, hereby incorporated by reference, the use of a double-stranded binding agent such as RNAseH may allow the reaction to proceed even at temperatures above the Tm of the primary probe:target hybridization complex. Accordingly, the addition of scissile probe to the target can be done either first, and then the cleavage agent or cleavage conditions introduced, or the probes may be added in the presence of the cleavage agent or conditions. [0174]
  • The cleavage conditions result in the separation of the two (or more) probe sequences of the primary scissile probe. As a result, the shorter probe sequences will no longer remain hybridized to the target sequence, and thus the hybridization complex will disassociate, leaving the target sequence intact. [0175]
  • The optimal temperature for carrying out the CPT reactions is generally from about 5° C. to about 25° C. below the melting temperatures of the probe:target hybridization complex. This provides for a rapid rate of hybridization and high degree of specificity for the target sequence. The Tm of any particular hybridization complex depends on salt concentration, G-C content, and length of the complex, as is known in the art and described herein. [0176]
  • During the reaction, as for the other amplification techniques herein, it may be necessary to suppress cleavage of the probe, as well as the target sequence, by nonspecific nucleases. Such nucleases are generally removed from the sample during the isolation of the DNA by heating or extraction procedures. A number of inhibitors of single-stranded nucleases such as vanadate, inhibitors it-ACE and RNAsin, a placental protein, do not affect the activity of RNAseH. This may not be necessary depending on the purity of the RNAseH and/or the target sample. [0177]
  • These steps are repeated by allowing the reaction to proceed for a period of time. The reaction is usually carried out for about 15 minutes to about 1 hour. Generally, each molecule of the target sequence will turnover between 100 and 1000 times in this period, depending on the length and sequence of the probe, the specific reaction conditions, and the cleavage method. For example, for each copy of the target sequence present in the test sample 100 to 1000 molecules will be cleaved by RNAseH. Higher levels of amplification can be obtained by allowing the reaction to proceed longer, or using secondary, tertiary, or quaternary probes, as is outlined herein. [0178]
  • Upon completion of the reaction, generally determined by time or amount of cleavage, the uncleaved scissile probes must be removed or neutralized prior to detection, such that the uncleaved probe does not bind to a detection probe, causing false positive signals. This may be done in a variety of ways, as is generally described below. [0179]
  • In a preferred embodiment, the separation is facilitated by the use of beads containing the primary probe. Thus, when the scissile probes are attached to beads, removal of the beads by filtration, centrifugation, the application of a magnetic field, electrostatic interactions for charged beads, adhesion, etc., results in the removal of the uncleaved probes. [0180]
  • In a preferred embodiment, the separation is based on strong acid precipitation. This is useful to separate long (generally greater than 50 nucleotides) from smaller fragments (generally about 10 nucleotides). The introduction of a strong acid such as trichloroacetic acid into the solution causes the longer probe to precipitate, while the smaller cleaved fragments remain in solution. The solution can be centrifuged or filtered to remove the precipitate, and the cleaved probe sequences can be quantitated. [0181]
  • In a preferred embodiment, the scissile probe contains both a detectable label and an affinity binding ligand or moiety, such that an affinity support is used to carry out the separation. In this embodiment, it is important that the detectable label used for detection is not on the same probe sequence that contains the affinity moiety, such that removal of the uncleaved probe, and the cleaved probe containing the affinity moiety, does not remove all the detectable labels. Alternatively, the scissile probe may contain a capture tag; the binding partner of the capture tag is attached to a solid support such as glass beads, latex beads, dextrans, etc. and used to pull out the uncleaved probes, as is known in the art. The cleaved probe sequences, which do not contain the capture tag, remain in solution and then can be detected as outlined below. [0182]
  • In a preferred embodiment, similar to the above embodiment, a separation sequence of nucleic acid is included in the scissile probe, which is not cleaved during the reaction. A nucleic acid complementary to the separation sequence is attached to a solid support such as a bead and serves as a catcher sequence. Preferably, the separation sequence is added to the scissile probes, and is not recognized by the target sequence, such that a generalized catcher sequence may be utilized in a variety of assays. [0183]
  • After removal of the uncleaved probe, as required, detection proceeds via the addition of the cleaved probe sequences to the array compositions, as outlined below. In general, the cleaved probe is bound to a capture probe, either directly or indirectly, and the label is detected. In a preferred embodiment, no higher order probes are used, and detection is based on the probe sequence(s) of the primary primer. In a preferred embodiment, at least one, and preferably more, secondary probes (also referred to herein as secondary primers) are used; the secondary probes hybridize to the domains of the cleavage probes; etc. [0184]
  • Thus, CPT requires, again in no particular order, a first CPT primer comprising a first probe sequence, a scissile linkage and a second probe sequence; and a cleavage agent. [0185]
  • In this manner, CPT results in the generation of a large amount of cleaved primers, which then can be detected as outlined below. [0186]
  • Labeling Techniques [0187]
  • In general, either direct or indirect detection of the target products can be done. “Direct” detection as used in this context, as for the other amplification strategies outlined herein, requires the incorporation of a label, in this case a detectable label, preferably an optical label such as a fluorophore, into the target sequence, with detection proceeding as outlined below. In this embodiment, the label(s) may be incorporated in three ways: (1) the primers comprise the label(s), for example attached to the base, a ribose, a phosphate, or to analogous structures in a nucleic acid analog; (2) modified nucleosides are used that are modified at either the base or the ribose (or to analogous structures in a nucleic acid analog) with the label(s); these label-modified nucleosides are then converted to the triphosphate form and are incorporated into the newly synthesized strand by a polymerase; (3) modified nucleotides are used that comprise a functional group that can be used to add a detectable label; or (4) modified primers are used that comprise a functional group that can be used to add a detectable label. Any of these methods result in a newly synthesized strand that comprises labels, that can be directly detected as outlined below. [0188]
  • Thus, the modified strands comprise a detection label. By “detection label” or “detectable label” herein is meant a moiety that allows detection. This may be a primary label or a secondary label. [0189]
  • In a preferred embodiment, the detection label is a primary label. A primary label is one that can be directly detected, such as a fluorophore. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic, electrical, thermal labels; and c) colored or luminescent dyes. Labels can also include enzymes (horseradish peroxidase, etc.) and magnetic particles. Preferred labels include chromophores or phosphors but are preferably fluorescent dyes. Suitable dyes for use in the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, alexa dyes, phycoerythin, bodipy, and others described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference. [0190]
  • In a preferred embodiment, a secondary detectable label is used. Accordingly, detection labels may be primary labels (i.e. directly detectable) or secondary labels (indirectly detectable). A secondary label is one that is indirectly detected; for example, a secondary label can bind or react with a primary label for detection, or may allow the separation of the compound comprising the secondary label from unlabeled materials, etc. Secondary labels find particular use in systems requiring separation of labeled and unlabeled probes, such as SBE reactions. Secondary labels include, but are not limited to, one of a binding partner pair; chemically modifiable moieties; nuclease inhibitors, etc. [0191]
  • In a preferred embodiment, the secondary label is a binding partner pair. For example, the label may be a hapten or antigen, which will bind its binding partner. In a preferred embodiment, the binding partner can be attached to a solid support to allow separation of extended and non-extended primers. For example, suitable binding partner pairs include, but are not limited to: antigens (such as proteins (including peptides)) and antibodies (including fragments thereof (FAbs, etc.)); proteins and small molecules, including biotin/streptavidin; enzymes and substrates or inhibitors; other protein-protein interacting pairs; receptor-ligands; and carbohydrates and their binding partners. Nucleic acid-nucleic acid binding proteins pairs are also useful. In general, the smaller of the pair is attached to the NTP for incorporation into the extension primer. [0192]
  • In a preferred embodiment, the binding partner pair comprises biotin or imino-biotin and streptavidin. Imino-biotin is particularly preferred as imino-biotin disassociates from streptavidin in pH 4.0 buffer while biotin requires harsh denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or 90% formamide at 95° C.). [0193]
  • In a preferred embodiment, the binding partner pair comprises a primary detection label (for example, attached to the NTP and therefore to the extended primer) and an antibody that will specifically bind to the primary detection label. By “specifically bind” herein is meant that the partners bind with specificity sufficient to differentiate between the pair and other components or contaminants of the system. The binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding. In some embodiments, the dissociation constants of the pair will be less than about 10[0194] −4-10−6 M−1, with less than about 10−5 to 10−9 M−1 being preferred and less than about 10−7-10−9 M−1 being particularly preferred.
  • For removal of unextended primers, it is preferred that the other half of the binding pair is attached to a solid support. In this embodiment, the solid support may be any as described herein for substrates and microspheres, and the form is preferably microspheres as well; for example, a preferred embodiment utilizes magnetic beads that can be easily introduced to the sample and easily removed, although any affinity chromatography formats may be used as well. Standard methods are used to attach the binding partner to the solid support, and can include direct or indirect attachment methods. For example, biotin labeled antibodies to fluorophores can be attached to streptavidin coated magnetic beads. [0195]
  • Thus, in this embodiment, the extended primers comprise a binding partner that is contacted with its binding partner under conditions wherein the extended primers are separated from the unextended primers. These extended primers can then be added to the array comprising capture probes as described herein. [0196]
  • In a preferred embodiment, the secondary label is a chemically modifiable moiety. In this embodiment, labels comprising reactive functional groups are incorporated into the nucleic acid. The functional group can then be subsequently labeled with a primary label. Suitable functional groups include, but are not limited to, amino groups, carboxy groups, maleimide groups, oxo groups and thiol groups, with amino groups and thiol groups being particularly preferred. [0197]
  • Removal of Unextended Primers [0198]
  • In a preferred embodiment, it is desirable to remove the unextended or unreacted primers from the assay mixture, and particularly from the array, as unextended primers will compete with the extended (labeled) primers in binding to capture probes, thereby diminishing the signal. The concentration of the unextended primers relative to the extended primer may be relatively high, since a large excess of primer is usually required to generate efficient primer annealing. Accordingly, a number of different techniques may be used to facilitate the removal of unextended primers. While the discussion below applies specifically to SBE, these techniques may be used in any of the methods described herein. [0199]
  • In a preferred embodiment, the NTPs (or, in the case of other methods, one or more of the probes) comprise a secondary detectable label that can be used to separate extended and non-extended primers. As outlined above, detection labels may be primary labels (i.e. directly detectable) or secondary labels (indirectly detectable). A secondary label is one that is indirectly detected; for example, a secondary label can bind or react with a primary label for detection, or may allow the separation of the compound comprising the secondary label from unlabeled materials, etc. Secondary labels find particular use in systems requiring separation of labeled and unlabeled probes, such as SBE, OLA, invasive cleavage, etc. reactions; in addition, these techniques may be used with many of the other techniques described herein. Secondary labels include, but are not limited to, one of a binding partner pair; chemically modifiable moieties; nuclease inhibitors, etc. [0200]
  • In a preferred embodiment, the secondary label is a binding partner pair. For example, the label may be a hapten or antigen, Which will bind its binding partner (generally attached to a solid support) and thus allow separation of extended and non-extended primers. For example, suitable binding partner pairs include, but are not limited to: antigens (such as proteins (including peptides)) and antibodies (including fragments thereof (FAbs, etc.)); proteins and small molecules, including biotin/streptavidin and digoxygenin and antibodies; enzymes and substrates or inhibitors; other protein-protein interacting pairs; receptor-ligands; and carbohydrates and their binding partners, are also suitable binding pairs. Nucleic acid-nucleic acid binding proteins pairs are also useful. In general, the smaller of the pair is attached to the NTP (or the probe) for incorporation into the extension primer. [0201]
  • In a preferred embodiment, the binding partner pair comprises biotin or imino-biotin and streptavidin. Imino-biotin is particularly preferred when the methods require the later separation of the pair, as imino-biotin disassociates from streptavidin in pH 4.0 buffer while biotin requires harsh denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or 90% formamide at 95° C.). [0202]
  • In addition, the use of streptavidin/biotin systems can be used to separate unreacted and reacted probes (for example in SBE, invasive cleavage, etc.). For example, the addition of streptavidin to a nucleic acid greatly increases its size, as well as changes its physical properties, to allow more efficient separation techniques. For example, the mixtures can be size fractionated by exclusion chromatography, affinity chromatography, filtration or differential precipitation. Alternatively, an 3′ exonuclease may be added to a mixture of 3′ labeled biotin/streptavidin; only the unreacted oligonucleotides will be degraded. Following exonuclease treatment, the exonuclease and the streptavidin can be degraded using a protease such as proteinase K. The surviving nucleic acids (i.e. those that were biotinylated) are then hybridized to the array. [0203]
  • In a preferred embodiment, the binding partner pair comprises a primary detection label (attached to the NTP and therefore to the extended primer) and an antibody that will specifically bind to the primary detection label. By “specifically bind” herein is meant that the partners bind with specificity sufficient to differentiate between the pair and other components or contaminants of the system. The binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding. In some embodiments, the dissociation constants of the pair will be less than about 10[0204] −4-10−6 M−1, with less than about 10−5 to 10−9 M−1 being preferred and less than about 10−7-10−9 M−1 being particularly preferred.
  • In this embodiment, it is preferred that the other half of the binding pair is attached to a solid support. In this embodiment, the solid support may be any as described herein for substrates and microspheres, and the form is preferably microspheres as well; for example, a preferred embodiment utilizes magnetic beads that can be easily introduced to the sample and easily removed, although any affinity chromatography formats may be used as well. Standard methods are used to attach the binding partner to the solid support, and can include direct or indirect attachment methods. For example, biotin labeled antibodies to fluorophores can be attached to streptavidin coated magnetic beads. [0205]
  • Thus, in this embodiment, the extended primers comprise a binding member that is contacted with its binding partner under conditions wherein the extended primers are separated from the unextended primers. These extended primers can then be added to the array comprising capture probes as described herein. [0206]
  • In a preferred embodiment, the secondary label is a chemically modifiable moiety. In this embodiment, labels comprising reactive functional groups are incorporated into the nucleic acid. [0207]
  • In a preferred embodiment, the secondary label is a nuclease inhibitor. In this embodiment, the chain-terminating NTPs are chosen to render extended primers resistant to nucleases, such as 3′-exonucleases. Addition of an exonuclease will digest the non-extended primers leaving only the extended primers to bind to the capture probes on the array. This may also be done with OLA, wherein the ligated probe will be protected but the unprotected ligation probe will be digested. [0208]
  • In this embodiment, suitable 3′-exonucleases include, but are not limited to, exo I, exo III, exo VII, etc. [0209]
  • Sandwich Assay Techniques [0210]
  • In a preferred embodiment, the signal amplification technique is a “sandwich” assay, as is generally described in U.S. Ser. No. 60/073,011 and in U.S. Pat. Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporated by reference. Although sandwich assays do not result in the alteration of primers, sandwich assays can be considered signal amplification techniques since multiple signals (i.e. label probes) are bound to a single target, resulting in the amplification of the signal. Sandwich assays may be used when the target sequence does not contain a label; or when adapters are used, as outlined below. [0211]
  • As discussed herein, it should be noted that the sandwich assays can be used for the detection of primary target sequences (e.g. from a patient sample), or as a method to detect the product of an amplification reaction as outlined above; thus for example, any of the newly synthesized strands outlined above, for example using PCR, LCR, NASBA, SDA, etc., may be used as the “target sequence” in a sandwich assay. [0212]
  • As will be appreciated by those in the art, the systems of the invention may take on a large number of different configurations. In general, there are three types of systems that can be used: (1) “non-sandwich” systems (also referred to herein as “direct” detection) in which the target sequence itself is labeled with detectable labels (again, either because the primers comprise labels or due to the incorporation of labels into the newly synthesized strand); (2) systems in which label probes directly bind to the target sequences; and (3) systems in which label probes are indirectly bound to the target sequences, for example through the use of amplifier probes. [0213]
  • The anchoring of the target sequence to the bead is done through the use of capture probes and optionally either capture extender probes (sometimes referred to as “adapter sequences” herein). When only capture probes are utilized, it is necessary to have unique capture probes for each target sequence; that is, the surface must be customized to contain unique capture probes; e.g. each bead comprises a different capture probe. Alternatively, capture extender probes may be used, that allow a “universal” surface, i.e. a surface containing a single type of capture probe that can be used to detect any target sequence. “Capture extender” probes have a first portion that will hybridize to all or part of the capture probe, and a second portion that will hybridize to a first portion of the target sequence. This then allows the generation of customized soluble probes, which as will be appreciated by those in the art is generally simpler and less costly. As shown herein, two capture extender probes may be used. This has generally been done to stabilize assay complexes for example when the target sequence is large, or when large amplifier probes (particularly branched or dendrimer amplifier probes) are used. [0214]
  • Detection of the amplification reactions of the invention, including the direct detection of amplification products and indirect detection utilizing label probes (i.e. sandwich assays), is preferably done by detecting assay complexes comprising detectable labels, which can be attached to the assay complex in a variety of ways, as is more fully described below. [0215]
  • Once the target sequence has preferably been anchored to the array, an amplifier probe is hybridized to the target sequence, either directly, or through the use of one or more label extender probes, which serves to allow “generic” amplifier probes to be made. As for all the steps outlined herein, this may be done simultaneously with capturing, or sequentially. Preferably, the amplifier probe contains a multiplicity of amplification sequences, although in some embodiments, as described below, the amplifier probe may contain only a single amplification sequence, or at least two amplification sequences. The amplifier probe may take on a number of different forms; either a branched conformation, a dendrimer conformation, or a linear “string” of amplification sequences. Label probes comprising detectable labels (preferably but not required to be fluorophores) then hybridize to the amplification sequences (or in some cases the label probes hybridize directly to the target sequence), and the labels detected, as is more fully outlined below. [0216]
  • Accordingly, the present invention provides compositions comprising an amplifier probe. By “amplifier probe” or “nucleic acid multimer” or “amplification multimer” or grammatical equivalents herein is meant a nucleic acid probe that is used to facilitate signal amplification. Amplifier probes comprise at least a first single-stranded nucleic acid probe sequence, as defined below, and at least one single-stranded nucleic acid amplification sequence, with a multiplicity of amplification sequences being preferred. [0217]
  • Amplifier probes comprise a first probe sequence that is used, either directly or indirectly, to hybridize to the target sequence. That is, the amplifier probe itself may have a first probe sequence that is substantially complementary to the target sequence, or it has a first probe sequence that is substantially complementary to a portion of an additional probe, in this case called a label extender probe, that has a first portion that is substantially complementary to the target sequence. In a preferred embodiment, the first probe sequence of the amplifier probe is substantially complementary to the target sequence. [0218]
  • In general, as for all the probes herein, the first probe sequence is of a length sufficient to give specificity and stability. Thus generally, the probe sequences of the invention that are designed to hybridize to another nucleic acid (i.e. probe sequences, amplification sequences, portions or domains of larger probes) are at least about 5 nucleosides long, with at least about 10 being preferred and at least about 15 being especially preferred. [0219]
  • In a preferred embodiment, several different amplifier probes are used, each with first probe sequences that will hybridize to a different portion of the target sequence. That is, there is more than one level of amplification; the amplifier probe provides an amplification of signal due to a multiplicity of labelling events, and several different amplifier probes, each with this multiplicity of labels, for each target sequence is used. Thus, preferred embodiments utilize at least two different pools of amplifier probes, each pool having a different probe sequence for hybridization to different portions of the target sequence; the only real limitation on the number of different amplifier probes will be the length of the original target sequence. In addition, it is also possible that the different amplifier probes contain different amplification sequences, although this is generally not preferred. [0220]
  • In a preferred embodiment, the amplifier probe does not hybridize to the sample target sequence directly, but instead hybridizes to a first portion of a label extender probe. This is particularly useful to allow the use of “generic” amplifier probes, that is, amplifier probes that can be used with a variety of different targets. This may be desirable since several of the amplifier probes require special synthesis techniques. Thus, the addition of a relatively short probe as a label extender probe is preferred. Thus, the first probe sequence of the amplifier probe is substantially complementary to a first portion or domain of a first label extender single-stranded nucleic acid probe. The label extender probe also contains a second portion or domain that is substantially complementary to a portion of the target sequence. Both of these portions are preferably at least about 10 to about 50 nucleotides in length, with a range of about 15 to about 30 being preferred. The terms “first” and “second” are not meant to confer an orientation of the sequences with respect to the 5′-3′ orientation of the target or probe sequences. For example, assuming a 5′-3′ orientation of the complementary target sequence, the first portion may be located either 5′ to the second portion, or 3′ to the second portion. For convenience herein, the order of probe sequences are generally shown from left to right. [0221]
  • In a preferred embodiment, more than one label extender probe-amplifier probe pair may be used, that is, n is more than 1. That is, a plurality of label extender probes may be used, each with a portion that is substantially complementary to a different portion of the target sequence; this can serve as another level of amplification. Thus, a preferred embodiment utilizes pools of at least two label extender probes, with the upper limit being set by the length of the target sequence. [0222]
  • In a preferred embodiment, more than one label extender probe is used with a single amplifier probe to reduce non-specific binding, as is generally outlined in U.S. Pat. No. 5,681,697, incorporated by reference herein. In this embodiment, a first portion of the first label extender probe hybridizes to a first portion of the target sequence, and the second portion of the first label extender probe hybridizes to a first probe sequence of the amplifier probe. A first portion of the second label extender probe hybridizes to a second portion of the target sequence, and the second portion of the second label extender probe hybridizes to a second probe sequence of the amplifier probe. These form structures sometimes referred to as “cruciform” structures or configurations, and are generally done to confer stability when large branched or dendrimeric amplifier probes are used. [0223]
  • In addition, as will be appreciated by those in the art, the label extender probes may interact with a preamplifier probe, described below, rather than the amplifier probe directly. [0224]
  • Similarly, as outlined above, a preferred embodiment utilizes several different amplifier probes, each with first probe sequences that will hybridize to a different portion of the label extender probe. In addition, as outlined above, it is also possible that the different amplifier probes contain different amplification sequences, although this is generally not preferred. [0225]
  • In addition to the first probe sequence, the amplifier probe also comprises at least one amplification sequence. An “amplification sequence” or “amplification segment” or grammatical equivalents herein is meant a sequence that is used, either directly or indirectly, to bind to a first portion of a label probe as is more fully described below. Preferably, the amplifier probe comprises a multiplicity of amplification sequences, with from about 3 to about 1000 being preferred, from about 10 to about 100 being particularly preferred, and about 50 being especially preferred. In some cases, for example when linear amplifier probes are used, from 1 to about 20 is preferred with from about 5 to about 10 being particularly preferred. [0226]
  • The amplification sequences may be linked to each other in a variety of ways, as will be appreciated by those in the art. They may be covalently linked directly to each other, or to intervening sequences or chemical moieties, through nucleic acid linkages such as phosphodiester bonds, PNA bonds, etc., or through interposed linking agents such amino acid, carbohydrate or polyol bridges, or through other cross-linking agents or binding partners. The site(s) of linkage may be at the ends of a segment, and/or at one or more internal nucleotides in the strand. In a preferred embodiment, the amplification sequences are attached via nucleic acid linkages. [0227]
  • In a preferred embodiment, branched amplifier probes are used, as are generally described in U.S. Pat. No. 5,124,246, hereby incorporated by reference. Branched amplifier probes may take on “fork-like” or “comb-like” conformations. “Fork-like” branched amplifier probes generally have three or more oligonucleotide segments emanating from a point of origin to form a branched structure. The point of origin may be another nucleotide segment or a multifunctional molecule to which at least three segments can be covalently or tightly bound. “Comb-like” branched amplifier probes have a linear backbone with a multiplicity of sidechain oligonucleotides extending from the backbone. In either conformation, the pendant segments will normally depend from a modified nucleotide or other organic moiety having the appropriate functional groups for attachment of oligonucleotides. Furthermore, in either conformation, a large number of amplification sequences are available for binding, either directly or indirectly, to detection probes. In general, these structures are made as is known in the art, using modified multifunctional nucleotides, as is described in U.S. Pat. Nos. 5,635,352 and 5,124,246, among others. [0228]
  • In a preferred embodiment, dendrimer amplifier probes are used, as are generally described in U.S. Pat. No. 5,175,270, hereby expressly incorporated by reference. Dendrimeric amplifier probes have amplification sequences that are attached via hybridization, and thus have portions of double-stranded nucleic acid as a component of their structure. The outer surface of the dendrimer amplifier probe has a multiplicity of amplification sequences. [0229]
  • In a preferred embodiment, linear amplifier probes are used, that have individual amplification sequences linked end-to-end either directly or with short intervening sequences to form a polymer. As with the other amplifier configurations, there may be additional sequences or moieties between the amplification sequences. In one embodiment, the linear amplifier probe has a single amplification sequence. [0230]
  • In addition, the amplifier probe may be totally linear, totally branched, totally dendrimeric, or any combination thereof. [0231]
  • The amplification sequences of the amplifier probe are used, either directly or indirectly, to bind to a label probe to allow detection. In a preferred embodiment, the amplification sequences of the amplifier probe are substantially complementary to a first portion of a label probe. Alternatively, amplifier extender probes are used, that have a first portion that binds to the amplification sequence and a second portion that binds to the first portion of the label probe. [0232]
  • In addition, the compositions of the invention may include “preamplifier” molecules, which serves a bridging moiety between the label extender molecules and the amplifier probes. In this way, more amplifier and thus more labels are ultimately bound to the detection probes. Preamplifier molecules may be either linear or branched, and typically contain in the range of about 30-3000 nucleotides. [0233]
  • Thus, label probes are either substantially complementary to an amplification sequence or to a portion of the target sequence. [0234]
  • Detection of the amplification reactions of the invention, including the direct detection of amplification products and indirect detection utilizing label probes (i.e. sandwich assays), is done by detecting assay complexes comprising labels. [0235]
  • Arrays [0236]
  • Detection of the amplified products described above preferably employs arrays, as defined herein. The arrays are preferably high density arrays that can allow simultaneous analysis, i.e. parallel rather than serial processing, on a number of samples. This is preferably done by forming an “array of arrays”, i.e. a composite array comprising a plurality of individual arrays, that is configured to allow processing of multiple samples, as is generally outlined in U.S. Ser. No. 09/256,943, hereby expressly incorporated by reference. For example, each individual array is present within each well of a microtiter plate. Thus, depending on the size of the microtiter plate and the size of the individual array, very high numbers of assays can be run simultaneously; for example, using individual arrays of 2,000 and a 96 well microtiter plate, 192,000 experiments can be done at once; the same arrays in a 384 microtiter plate yields 768,000 simultaneous experiments, and a 1536 microtiter plate gives 3,072,000 experiments. [0237]
  • Generally, the array of array compositions of the invention can be configured in several ways. In a preferred embodiment, as is more fully outlined below, a “one component” system is used. That is, a first substrate comprising a plurality of assay locations (sometimes also referred to herein as “assay wells”), such as a microtiter plate, is configured such that each assay location contains an individual array. That is, the assay location and the array location are the same. For example, the plastic material of the microtiter plate can be formed to contain a plurality of “bead wells” in the bottom of each of the assay wells. Beads containing the capture probes of the invention can then be loaded into the bead wells in each assay location as is more fully described below. [0238]
  • Alternatively, a “two component” system can be used. In this embodiment, the individual arrays are formed on a second substrate, which then can be fitted or “dipped” into the first microtiter plate substrate. A preferred embodiment utilizes fiber optic bundles as the individual arrays, generally with “bead wells” etched into one surface of each individual fiber, such that the beads containing the capture probes are loaded onto the end of the fiber optic bundle. The composite array thus comprises a number of individual arrays that are configured to fit within the wells of a microtiter plate. [0239]
  • The present invention is generally based on previous work comprising a bead-based analytic chemistry system in which beads, also termed microspheres, carrying different chemical functionalities are distributed on a substrate comprising a patterned surface of discrete sites that can bind the individual microspheres. The beads are generally put onto the substrate randomly, and thus several different methodologies can be used to “decode” the arrays. In one embodiment, unique optical signatures are incorporated into the beads, generally fluorescent dyes, that could be used to identify the chemical functionality on any particular bead. This allows the synthesis of the nucleic acids to be divorced from their placement on an array, i.e. the capture probes may be synthesized on the beads, and then the beads are randomly distributed on a patterned surface. Since the beads are first coded with an optical signature, this means that the array can later be “decoded”, i.e. after the array is made, a correlation of the location of an individual site on the array with the probe at that particular site can be made. This means that the beads may be randomly distributed on the array, a fast and inexpensive process as compared to either the in situ synthesis or spotting techniques of the prior art. These methods are generally outlined in PCTs US98/05025 and US99/14387 and U.S. Ser. Nos. 08/818,199 and 09/151,877, all of which are expressly incorporated herein by reference. [0240]
  • However, the drawback to these methods is that for a very high density array, the system requires a large number of different optical signatures, which may be difficult or time-consuming to utilize. Accordingly, the present invention also provides several improvements over these methods, generally directed to methods of coding and decoding the arrays. That is, as will be appreciated by those in the art, the placement of the probes is generally random, and thus a coding/decoding system is required to identify the probes at each location in the array. This may be done in a variety of ways, as is more fully outlined below, and generally includes: a) the use a decoding binding ligand (DBL), generally directly labeled, that binds to either the capture probes or to identifier binding ligands (IBLs) attached to the beads; b) positional decoding, for example by either targeting the placement of beads (for example by using photoactivatible or photocleavable moieties to allow the selective addition of beads to particular locations), or by using either sub-bundles or selective loading of the sites, as are more fully outlined below; c) selective decoding, wherein only those beads that bind to a target are decoded; or d) combinations of any of these. In some cases, as is more fully outlined below, this decoding may occur for all the beads, or only for those that bind a particular target analyte. Similarly, this may occur either prior to or after addition of a target analyte. [0241]
  • Once the identity (i.e. the actual agent) and location of each microsphere in the array has been fixed, the array is exposed to samples containing the target sequences, such as, the products of amplification reactions described above, although as outlined below, this can be done prior to or during the analysis as well. The target sequences will bind to the capture probes as is more fully outlined below, and results (in the case of optical labels) in a change in the optical signal of a particular bead. [0242]
  • In the present invention, “decoding” can use optical signatures, decoding binding ligands that are added during a decoding step, or a combination of these methods. The decoding binding ligands will bind either to a distinct identifier binding ligand partner that is placed on the beads, or to the capture probes, with the latter being preferred. The decoding binding ligands are either directly or indirectly labeled, and thus decoding occurs by detecting the presence of the label. By using pools of decoding binding ligands in a sequential fashion, it is possible to greatly minimize the number of required decoding steps. [0243]
  • Accordingly, the present invention provides composite array compositions comprising at least a first substrate with a surface comprising a plurality of assay locations. By “array” herein is meant a plurality of candidate agents in an array format; the size of the array will depend on the composition and end use of the array. Arrays containing from about 2 different probes (i.e. different beads) to many millions can be made, with very large fiber optic arrays being possible. Generally, the array will comprise from two to as many as a billion or more per square cm, depending on the size of the beads and the substrate, as well as the end use of the array, thus very high density, high density, moderate density, low density and very low density arrays may be made. Preferred ranges for very high density arrays are from about 10,000,000 to about 2,000,000,000, (with all numbers being per square centimeter) with from about 100,000,000 to about 1,000,000,000 being preferred. High density arrays range about 100,000 to about 10,000,000, with from about 1,000,000 to about 5,000,000 being particularly preferred. Moderate density arrays range from about 10,000 to about 100,000 being particularly preferred, and from about 20,000 to about 50,000 being especially preferred. Low density arrays are generally less than 10,000, with from about 1,000 to about 5,000 being preferred. Very low density arrays are less than 1,000, with from about 10 to about 1000 being preferred, and from about 100 to about 500 being particularly preferred. In some embodiments, the compositions of the invention may not be in array format; that is, for some embodiments, compositions comprising a single bioactive agent may be made as well. In addition, in some arrays, multiple substrates may be used, either of different or identical compositions. Thus for example, large arrays may comprise a plurality of smaller substrates. [0244]
  • In addition, one advantage of the present compositions is that particularly through the use of fiber optic technology, extremely high density arrays can be made. Thus for example, because beads of 200 μm or less (with beads of 200 nm possible) can be used, and very small fibers are known, it is possible to have as many as 250,000 or more (in some instances, 1 million) different fibers and beads in a 1 mm[0245] 2 fiber optic bundle, with densities of greater than 15,000,000 individual beads and fibers (again, in some instances as many as 25-50 million) per 0.5 cm2 obtainable.
  • By “composite array” or “combination array” or grammatical equivalents herein is meant a plurality of individual arrays, as outlined above. Generally the number of individual arrays is set by the size of the microtiter plate used; thus, 96 well, 384 well and 1536 well microtiter plates utilize composite arrays comprising 96, 384 and 1536 individual arrays, although as will be appreciated by those in the art, not each microtiter well need contain an individual array. It should be noted that the composite arrays can comprise individual arrays that are identical, similar or different. That is, in some embodiments, it may be desirable to do the same 2,000 assays on 96 different samples; alternatively, doing 192,000 experiments on the same sample (i.e. the same sample in each of the 96 wells) may be desirable. Alternatively, each row or column of the composite array could be the same, for redundancy/quality control. As will be appreciated by those in the art, there are a variety of ways to configure the system. In addition, the random nature of the arrays may mean that the same population of beads may be added to two different surfaces, resulting in substantially similar but perhaps not identical arrays. [0246]
  • By “substrate” or “solid support” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of beads and is amenable to at least one detection method. As will be appreciated by those in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers. In general, the substrates allow optical detection and do not themselves appreciably fluorescese. [0247]
  • Generally the substrate is flat (planar), although as will be appreciated by those in the art, other configurations of substrates may be used as well; for example, three dimensional configurations can be used, for example by embedding the beads in a porous block of plastic that allows sample access to the beads and using a confocal microscope for detection. Similarly, the beads may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume. Preferred substrates include optical fiber bundles as discussed below, and flat planar substrates such as glass, polystyrene and other plastics and acrylics. [0248]
  • The first substrate comprises a surface comprising a plurality of assay locations, i.e. the location where the assay for the detection of a target analyte will occur. The assay locations are generally physically separated from each other, for example as assay wells in a microtiter plate, although other configurations (hydrophobicity/hydrophilicity, etc.) can be used to separate the assay locations. [0249]
  • In a preferred embodiment, the second substrate is an optical fiber bundle or array, as is generally described in U.S. Ser. Nos. 08/944,850 and 08/519,062, PCT US98/05025, and PCT US98/09163, all of which are expressly incorporated herein by reference. Preferred embodiments utilize preformed unitary fiber optic arrays. By “preformed unitary fiber optic array” herein is meant an array of discrete individual fiber optic strands that are co-axially disposed and joined along their lengths. The fiber strands are generally individually clad. However, one thing that distinguished a preformed unitary array from other fiber optic formats is that the fibers are not individually physically manipulatable; that is, one strand generally cannot be physically separated at any point along its length from another fiber strand. [0250]
  • In a preferred embodiment, the array comprises a plurality of discrete sites. Thus, in the former case, the assay location is the same as the array location, as described herein. In the latter case, the array location is fitted into the assay location separately. In these embodiments, at least one surface of the substrate is modified to contain discrete, individual sites for later association of microspheres. These sites may comprise physically altered sites, i.e. physical configurations such as wells or small depressions in the substrate that can retain the beads, such that a microsphere can rest in the well, or the use of other forces (magnetic or compressive), or chemically altered or active sites, such as chemically functionalized sites, electrostatically altered sites, hydrophobically/hydrophilically functionalized sites, spots of adhesive, etc. [0251]
  • The sites may be a pattern, i.e. a regular design or configuration, or randomly distributed. A preferred embodiment utilizes a regular pattern of sites such that the sites may be addressed in the X-Y coordinate plane. “Pattern” in this sense includes a repeating unit cell, preferably one that allows a high density of beads on the substrate. However, it should be noted that these sites may not be discrete sites. That is, it is possible to use a uniform surface of adhesive or chemical functionalities, for example, that allows the attachment of beads at any position. That is, the surface of the substrate is modified to allow attachment of the microspheres at individual sites, whether or not those sites are contiguous or non-contiguous with other sites. Thus, the surface of the substrate may be modified such that discrete sites are formed that can only have a single associated bead, or alternatively, the surface of the substrate is modified and beads may go down anywhere, but they end up at discrete sites. [0252]
  • In a preferred embodiment, the surface of the substrate is modified to contain wells, i.e. depressions in the surface of the substrate. This may be done as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques. As will be appreciated by those in the art, the technique used will depend on the composition and shape of the substrate. When the first substrate comprises both the assay locations and the individual arrays, a preferred method utilizes molding techniques that form the bead wells in the bottom of the assay wells in a microtiter plate. [0253]
  • In a preferred embodiment, physical alterations are made in a surface of the substrate to produce the sites. In a preferred embodiment, for example when the second substrate is a fiber optic bundle, the surface of the substrate is a terminal end of the fiber bundle, as is generally described in 08/818,199 and 09/151,877, both of which are hereby expressly incorporated by reference. In this embodiment, wells are made in a terminal or distal end of a fiber optic bundle comprising individual fibers. In this embodiment, the cores of the individual fibers are etched, with respect to the cladding, such that small wells or depressions are formed at one end of the fibers. The required depth of the wells will depend on the size of the beads to be added to the wells. [0254]
  • Generally in this embodiment, the microspheres are non-covalently associated in the wells, although the wells may additionally be chemically functionalized as is generally described below, cross-linking agents may be used, or a physical barrier may be used, i.e. a film or membrane over the beads. [0255]
  • In a preferred embodiment, the surface of the substrate is modified to contain chemically modified sites, that can be used to attach, either covalently or non-covalently, the microspheres of the invention to the discrete sites or locations on the substrate. “Chemically modified sites” in this context includes, but is not limited to, the addition of a pattern of chemical functional groups including amino groups, carboxy groups, oxo groups and thiol groups, that can be used to covalently attach microspheres, which generally also contain corresponding reactive functional groups; the addition of a pattern of adhesive that can be used to bind the microspheres (either by prior chemical functionalization for the addition of the adhesive or direct addition of the adhesive); the addition of a pattern of charged groups (similar to the chemical functionalities) for the electrostatic attachment of the microspheres, i.e. when the microspheres comprise charged groups opposite to the sites; the addition of a pattern of chemical functional groups that renders the sites differentially hydrophobic or hydrophilic, such that the addition of similarly hydrophobic or hydrophilic microspheres under suitable experimental conditions will result in association of the microspheres to the sites on the basis of hydroaffinity. For example, the use of hydrophobic sites with hydrophobic beads, in an aqueous system, drives the association of the beads preferentially onto the sites. As outlined above, “pattern” in this sense includes the use of a uniform treatment of the surface to allow attachment of the beads at discrete sites, as well as treatment of the surface resulting in discrete sites. As will be appreciated by those in the art, this may be accomplished in a variety of ways. [0256]
  • The compositions of the invention further comprise a population of microspheres. By “population” herein is meant a plurality of beads as outlined above for arrays. Within the population are separate subpopulations, which can be a single microsphere or multiple identical microspheres. That is, in some embodiments, as is more fully outlined below, the array may contain only a single bead for each bioactive agent; preferred embodiments utilize a plurality of beads of each type. [0257]
  • By “microspheres” or “beads” or “particles” or grammatical equivalents herein is meant small discrete particles. The composition of the beads will vary, depending on the class of bioactive agent and the method of synthesis. Suitable bead compositions include those used in peptide, nucleic acid and organic moiety synthesis, including, but not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sol, carbon graphited, titanium dioxide, latex or cross-linked dextrans such as Sepharose, cellulose, nylon, cross-linked micelles and teflon may all be used. “Microsphere Detection Guide” from Bangs Laboratories, Fishers IN is a helpful guide. [0258]
  • The beads need not be spherical; irregular particles may be used. In addition, the beads may be porous, thus increasing the surface area of the bead available for assay. The bead sizes range from nanometers, i.e. 100 nm, to millimeters, i.e. 1 mm, with beads from about 0.2 micron to about 200 microns being preferred, and from about 0.5 to about 5 micron being particularly preferred, although in some embodiments smaller beads may be used. [0259]
  • It should be noted that a key component of the invention is the use of a substrate/bead pairing that allows the association or attachment of the beads at discrete sites on the surface of the substrate, such that the beads do not move during the course of the assay. [0260]
  • Each microsphere comprises a capture probe although as will be appreciated by those in the art, there may be some microspheres which do not contain a capture probe, depending the on the synthetic methods. By “capture probe” or “capture nucleic acid” herein is meant a probe for the direct or indirect binding of the target sequence to a bead. By “nucleic acid” or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together, as described above. [0261]
  • In a preferred embodiment, each bead comprises a single type of capture probes, although a plurality of individual probes are preferably attached to each bead. Similarly, preferred embodiments utilize more than one microsphere containing a unique capture probe; that is, there is redundancy built into the system by the use of subpopulations of microspheres, each microsphere in the subpopulation containing the same probe. [0262]
  • As will be appreciated by those in the art, the probes may either be synthesized directly on the beads, or they may be made and then attached after synthesis. In a preferred embodiment, linkers are used to attach the probes to the beads, to allow both good attachment, sufficient flexibility to allow good interaction with the target sequence, and to avoid undesirable binding reactions. [0263]
  • In a preferred embodiment, the probes are synthesized directly on the beads. As is known in the art, many classes of chemical compounds are currently synthesized on solid supports, such as peptides, organic moieties, and nucleic acids. It is a relatively straightforward matter to adjust the current synthetic techniques to use beads. [0264]
  • In a preferred embodiment, the probes are synthesized first, and then covalently attached to the beads. As will be appreciated by those in the art, this will be done depending on the composition of the bioactive agents and the beads. The functionalization of solid support surfaces such as certain polymers with chemically reactive groups such as thiols, amines, carboxyls, etc. is generally known in the art. Accordingly, “blank” microspheres may be used that have surface chemistries that facilitate the attachment of the desired functionality by the user. Some examples of these surface chemistries for blank microspheres include, but are not limited to, amino groups including aliphatic and aromatic amines, carboxylic acids, aldehydes, amides, chloromethyl groups, hydrazide, hydroxyl groups, sulfonates and sulfates. [0265]
  • In some embodiments, the beads may additionally comprise an optical signature, that can be used to identify the bioactive agent; see for example U.S. Ser. Nos. 08/818,199 and 09/151,877, and PCT US98/05025, all of which are expressly incorporated herein by reference. [0266]
  • In some embodiments, the microspheres may additionally comprise identifier binding ligands for use in certain decoding systems. By “identifier binding ligands” or “IBLs” herein is meant a compound that will specifically bind a corresponding decoder binding ligand (DBL) to facilitate the elucidation of the identity of the bioactive agent attached to the bead. That is, the IBL and the corresponding DBL form a binding partner pair. By “specifically bind” herein is meant that the IBL binds its DBL with specificity sufficient to differentiate between the corresponding DBL and other DBLs (that is, DBLs for other IBLs), or other components or contaminants of the system. The binding should be sufficient to remain bound under the conditions of the decoding step, including wash steps to remove non-specific binding. In some embodiments, for example when the IBLs and corresponding DBLs are proteins or nucleic acids, the dissociation constants of the IBL to its DBL will be less than about 10[0267] −4-10−6 M−1, with less than about 10−5 to 10−9 M−1 being preferred and less than about 10−7-10−9 M−1 being particularly preferred.
  • IBL-DBL binding pairs are known or can be readily found using known techniques. For example, when the IBL is a protein, the DBLs include proteins (particularly including antibodies or fragments thereof (FAbs, etc.)) or small molecules, or vice versa (the IBL is an antibody and the DBL is a protein). By “protein” herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus “amino acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and norleucine are considered amino acids for the purposes of the invention. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations. Metal ion-metal ion ligands or chelators pairs are also useful. Antigen-antibody pairs, enzymes and substrates or inhibitors, other protein-protein interacting pairs, receptor-ligands, complementary nucleic acids, and carbohydrates and their binding partners are also suitable binding pairs. Nucleic acid-nucleic acid binding proteins pairs are also useful. Similarly, as is generally described in U.S. Pat. Nos. 5,270,163, 5,475,096, 5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and related patents, hereby incorporated by reference, nucleic acid “aptomers” can be developed for binding to virtually any target; such a aptomer-target pair can be used as the IBL-DBL pair. Similarly, there is a wide body of literature relating to the development of binding pairs based on combinatorial chemistry methods. [0268]
  • In a preferred embodiment, the IBL is a molecule whose color or luminescence properties change in the presence of a selectively-binding DBL. For example, the IBL may be a fluorescent pH indicator whose emission intensity changes with pH. Similarly, the IBL may be a fluorescent ion indicator, whose emission properties change with ion concentration. [0269]
  • Alternatively, the IBL is a molecule whose color or luminescence properties change in the presence of various solvents. For example, the IBL may be a fluorescent molecule such as an ethidium salt whose fluorescence intensity increases in hydrophobic environments. Similarly, the IBL may be a derivative of fluorescein whose color changes between aqueous and nonpolar solvents. [0270]
  • In one embodiment, the DBL may be attached to a bead, i.e. a “decoder bead”, that may carry a label such as a fluorophore. [0271]
  • In a preferred embodiment, the IBL-DBL pair comprise substantially complementary single-stranded nucleic acids. In this embodiment, the binding ligands can be referred to as “identifier probes” and “decoder probes”. Generally, the identifier and decoder probes range from about 4 basepairs in length to about 1000, with from about 6 to about 100 being preferred, and from about 8 to about 40 being particularly preferred. What is important is that the probes are long enough to be specific, i.e. to distinguish between different IBL-DBL pairs, yet short enough to allow both a) dissociation, if necessary, under suitable experimental conditions, and b) efficient hybridization. [0272]
  • In a preferred embodiment, as is more fully outlined below, the IBLs do not bind to DBLs. Rather, the IBLs are used as identifier moieties (“IMs”) that are identified directly, for example through the use of mass spectroscopy. [0273]
  • In a preferred embodiment, the microspheres may contain an optical signature. That is, as outlined in U.S. Ser. Nos. 08/818,199 and 09/151,877, previous work had each subpopulation of microspheres comprising a unique optical signature or optical tag that is used to identify the unique capture probe of that subpopulation of microspheres; that is, decoding utilizes optical properties of the beads such that a bead comprising the unique optical signature may be distinguished from beads at other locations with different optical signatures. Thus the previous work assigned each probe a unique optical signature such that any microspheres comprising that probe are identifiable on the basis of the signature. These optical signatures comprised dyes, usually chromophores or fluorophores, that were entrapped or attached to the beads themselves. Diversity of optical signatures utilized different fluorochromes, different ratios of mixtures of fluorochromes, and different concentrations (intensities) of fluorochromes. [0274]
  • While generally, the present invention does not rely solely on the use of optical properties to decode the arrays, as will be appreciated by those in the art, it is possible in some embodiments to utilize optical signatures as an additional coding method, in conjunction with the present system. Thus, for example, as is more fully outlined below, the size of the array may be effectively increased while using a single set of decoding moieties in several ways, one of which is the use of optical signatures one some beads. Thus, for example, using one “set” of decoding molecules, the use of two populations of beads, one with an optical signature and one without, allows the effective doubling of the array size. The use of multiple optical signatures similarly increases the possible size of the array. [0275]
  • In a preferred embodiment, each subpopulation of beads comprises a plurality of different IBLs. By using a plurality of different IBLs to encode each probe, the number of possible unique codes is substantially increased. That is, by using one unique IBL per probe, the size of the array will be the number of unique IBLs (assuming no “reuse” occurs, as outlined below). However, by using a plurality of different IBLs per bead, n, the size of the array can be increased to 2[0276] n, when the presence or absence of each IBL is used as the indicator. For example, the assignment of 10 IBLs per bead generates a 10 bit binary code, where each bit can be designated as “1” (IBL is present) or “0” (IBL is absent). A 10 bit binary code has 210 possible variants. However, as is more fully discussed below, the size of the array may be further increased if another parameter is included such as concentration or intensity; thus for example, if two different concentrations of the IBL are used, then the array size increases as 3n. Thus, in this embodiment, each individual probe in the array is assigned a combination of IBLs, which can be added to the beads prior to the addition of the probe, after, or during the synthesis of the probe, i.e. simultaneous addition of IBLs and probes.
  • In some embodiment, the combination of different IBLs can be used to elucidate the sequence of the probe. [0277]
  • Thus, for example, using two different IBLs (IBL1 and IBL2), the first position of a nucleic acid can be elucidated: for example, adenosine can be represented by the presence of both IBL1 and IBL2; thymidine can be represented by the presence of IBL1 but not IBL2, cytosine can be represented by the presence of IBL2 but not IBL1, and guanosine can be represented by the absence of both. The second position of the nucleic acid can be done in a similar manner using IBL3 and IBL4; thus, the presence of IBL1, IBL2, IBL3 and IBL4 gives a sequence of AA; IBL1, IBL2, and IBL3 shows the sequence AT; IBL1, IBL3 and IBL4 gives the sequence TA, etc. The third position utilizes IBL5 and IBL6, etc. In this way, the use of 20 different identifiers can yield a unique code for every possible 10-mer. [0278]
  • In this way, a sort of “bar code” for each sequence can be constructed; the presence or absence of each distinct IBL will allow the identification of each probe. [0279]
  • In addition, the use of different concentrations or densities of IBLs allows a “reuse” of sorts. If, for example, the bead comprising a first agent has a 1×concentration of IBL, and a second bead comprising a second agent has a 10×concentration of IBL, using saturating concentrations of the corresponding labelled DBL allows the user to distinguish between the two beads. [0280]
  • Once the microspheres comprising the probes and the unique tags are generated, they are added to the substrate to form an array. In general, the methods of making the arrays and of decoding the arrays is done to maximize the number of different candidate agents that can be uniquely encoded. The compositions of the invention may be made in a variety of ways. In general, the arrays are made by adding a solution or slurry comprising the beads to a surface containing the sites for attachment of the beads. This may be done in a variety of buffers, including aqueous and organic solvents, and mixtures. The solvent can evaporate, and excess beads removed. [0281]
  • In a preferred embodiment, when non-covalent methods are used to associate the beads to the array, a novel method of loading the beads onto the array is used. This method comprises exposing the array to a solution of particles (including microspheres and cells) and then applying energy, e.g. agitating or vibrating the mixture. This results in an array comprising more tightly associated particles, as the agitation is done with sufficient energy to cause weakly-associated beads to fall off (or out, in the case of wells). These sites are then available to bind a different bead. In this way, beads that exhibit a high affinity for the sites are selected. Arrays made in this way have two main advantages as compared to a more static loading: first of all, a higher percentage of the sites can be filled easily, and secondly, the arrays thus loaded show a substantial decrease in bead loss during assays. Thus, in a preferred embodiment, these methods are used to generate arrays that have at least about 50% of the sites filled, with at least about 75% being preferred, and at least about 90% being particularly preferred. Similarly, arrays generated in this manner preferably lose less than about 20% of the beads during an assay, with less than about 10% being preferred and less than about 5% being particularly preferred. [0282]
  • In this embodiment, the substrate comprising the surface with the discrete sites is immersed into a solution comprising the particles (beads, cells, etc.). The surface may comprise wells, as is described herein, or other types of sites on a patterned surface such that there is a differential affinity for the sites. This differential affinity results in a competitive process, such that particles that will associate more tightly are selected. Preferably, the entire surface to be “loaded” with beads is in fluid contact with the solution. This solution is generally a slurry ranging from about 10,000:1 beads:solution (vol:vol) to 1:1. Generally, the solution can comprise any number of reagents, including aqueous buffers, organic solvents, salts, other reagent components, etc. In addition, the solution preferably comprises an excess of beads; that is, there are more beads than sites on the array. Preferred embodiments utilize two-fold to billion-fold excess of beads. [0283]
  • The immersion can mimic the assay conditions; for example, if the array is to be “dipped” from above into a microtiter plate comprising samples, this configuration can be repeated for the loading, thus minimizing the beads that are likely to fall out due to gravity. [0284]
  • Once the surface has been immersed, the substrate, the solution, or both are subjected to a competitive process, whereby the particles with lower affinity can be disassociated from the substrate and replaced by particles exhibiting a higher affinity to the site. This competitive process is done by the introduction of energy, in the form of heat, sonication, stirring or mixing, vibrating or agitating the solution or substrate, or both. [0285]
  • A preferred embodiment utilizes agitation or vibration. In general, the amount of manipulation of the substrate is minimized to prevent damage to the array; thus, preferred embodiments utilize the agitation of the solution rather than the array, although either will work. As will be appreciated by those in the art, this agitation can take on any number of forms, with a preferred embodiment utilizing microtiter plates comprising bead solutions being agitated using microtiter plate shakers. [0286]
  • The agitation proceeds for a period of time sufficient to load the array to a desired fill. Depending on the size and concentration of the beads and the size of the array, this time may range from about 1 second to days, with from about 1 minute to about 24 hours being preferred. [0287]
  • It should be noted that not all sites of an array may comprise a bead; that is, there may be some sites on the substrate surface which are empty. In addition, there may be some sites that contain more than one bead, although this is not preferred. [0288]
  • In some embodiments, for example when chemical attachment is done, it is possible to attach the beads in a non-random or ordered way. For example, using photoactivatible attachment linkers or photoactivatible adhesives or masks, selected sites on the array may be sequentially rendered suitable for attachment, such that defined populations of beads are laid down. [0289]
  • The arrays of the present invention are constructed such that information about the identity of the candidate agent is built into the array, such that the random deposition of the beads in the fiber wells can be “decoded” to allow identification of the candidate agent at all positions. This may be done in a variety of ways, and either before, during or after the use of the array to detect target molecules. [0290]
  • Thus, after the array is made, it is “decoded” in order to identify the location of one or more of the probes, i.e. each subpopulation of beads, on the substrate surface. [0291]
  • In a preferred embodiment, a selective decoding system is used. In this case, only those microspheres exhibiting a change in the optical signal as a result of the binding of a target analyte are decoded. This is commonly done when the number of “hits”, i.e. the number of sites to decode, is generally low. That is, the array is first scanned under experimental conditions in the absence of the target analytes. The sample containing the target analytes is added, and only those locations exhibiting a change in the optical signal are decoded. For example, the beads at either the positive or negative signal locations may be either selectively tagged or released from the array (for example through the use of photocleavable linkers), and subsequently sorted or enriched in a fluorescence-activated cell sorter (FACS). That is, either all the negative beads are released, and then the positive beads are either released or analyzed in situ, or alternatively all the positives are released and analyzed. Alternatively, the labels may comprise halogenated aromatic compounds, and detection of the label is done using for example gas chromatography, chemical tags, isotopic tags mass spectral tags. [0292]
  • As will be appreciated by those in the art, this may also be done in systems where the array is not decoded; i.e. there need not ever be a correlation of bead composition with location. In this embodiment, the beads are loaded on the array, and the assay is run. The “positives”, i.e. those beads displaying a change in the optical signal as is more fully outlined below, are then “marked” to distinguish or separate them from the “negative” beads. This can be done in several ways, preferably using fiber optic arrays. In a preferred embodiment, each bead contains a fluorescent dye. After the assay and the identification of the “positives” or “active beads”, light is shown down either only the positive fibers or only the negative fibers, generally in the presence of a light-activated reagent (typically dissolved oxygen). In the former case, all the active beads are photobleached. Thus, upon non-selective release of all the beads with subsequent sorting, for example using a fluorescence activated cell sorter (FACS) machine, the non-fluorescent active beads can be sorted from the fluorescent negative beads. Alternatively, when light is shown down the negative fibers, all the negatives are non-fluorescent and the the positives are fluorescent, and sorting can proceed. The characterization of the attached probe may be done directly, for example using mass spectroscopy. [0293]
  • Alternatively, the identification may occur through the use of identifier moieties (“IMs”), which are similar to IBLs but need not necessarily bind to DBLs. That is, rather than elucidate the structure of the capture probe directly, the composition of the IMs may serve as the identifier. Thus, for example, a specific combination of IMs can serve to code the bead, and be used to identify the agent on the bead upon release from the bead followed by subsequent analysis, for example using a gas chromatograph or mass spectroscope. [0294]
  • Alternatively, rather than having each bead contain a fluorescent dye, each bead comprises a non-fluorescent precursor to a fluorescent dye. For example, using photocleavable protecting groups, such as certain ortho-nitrobenzyl groups, on a fluorescent molecule, photoactivation of the fluorochrome can be done. After the assay, light is shown down again either the “positive” or the “negative” fibers, to distinquish these populations. The illuminated precursors are then chemically converted to a fluorescent dye. All the beads are then released from the array, with sorting, to form populations of fluorescent and non-fluorescent beads (either the positives and the negatives or vice versa). [0295]
  • In an alternate preferred embodiment, the sites of attachment of the beads (for example the wells) include a photopolymerizable reagent, or the photopolymerizable agent is added to the assembled array. After the test assay is run, light is shown down again either the “positive” or the “negative” fibers, to distinquish these populations. As a result of the irradiation, either all the positives or all the negatives are polymerized and trapped or bound to the sites, while the other population of beads can be released from the array. [0296]
  • In a preferred embodiment, the location of every capture probe is determined using decoder binding ligands (DBLs). As outlined above, DBLs are binding ligands that will either bind to identifier binding ligands, if present, or to the capture probes themselves. [0297]
  • In a preferred embodiment, as outlined above, the DBL binds to the IBL. [0298]
  • In a preferred embodiment, the capture probes are single-stranded nucleic acids and the DBL is a substantially complementary single-stranded nucleic acid that binds (hybridizes) to the capture probe, termed a decoder probe herein. A decoder probe that is substantially complementary to each candidate probe is made and used to decode the array. In this embodiment, the candidate probes and the decoder probes should be of sufficient length (and the decoding step run under suitable conditions) to allow specificity; i.e. each candidate probe binds to its corresponding decoder probe with sufficient specificity to allow the distinction of each candidate probe. [0299]
  • In a preferred embodiment, the DBLs are either directly or indirectly labeled. By “labeled” herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic, electrical, thermal; and c) colored or luminescent dyes; although labels include enzymes and particles such as magnetic particles as well. Preferred labels include luminescent labels. In a preferred embodiment, the DBL is directly labeled, that is, the DBL comprises a label. In an alternate embodiment, the DBL is indirectly labeled; that is, a labeling binding ligand (LBL) that will bind to the DBL is used. In this embodiment, the labeling binding ligand-DBL pair can be as described above for IBL-DBL pairs. [0300]
  • Accordingly, the identification of the location of the individual beads (or subpopulations of beads) is done using one or more decoding steps comprising a binding between the labeled DBL and either the IBL or the capture probe (i.e. a hybridization between the capture probe and the decoder probe). After decoding, the DBLs can be removed and the array can be used; however, in some circumstances, for example when the DBL binds to an IBL and not to the capture probe, the removal of the DBL is not required (although it may be desirable in some circumstances). In addition, as outlined herein, decoding may be done either before the array is used to in an assay, during the assay, or after the assay. [0301]
  • In one embodiment, a single decoding step is done. In this embodiment, each DBL is labeled with a unique label, such that the the number of unique tags is equal to or greater than the number of capture probe (although in some cases, “reuse” of the unique labels can be done, as described herein; similarly, minor variants of candidate probes can share the same decoder, if the variants are encoded in another dimension, i.e. in the bead size or label). For each capture probe or IBL, a DBL is made that will specifically bind to it and contains a unique tag, for example one or more fluorochromes. Thus, the identity of each DBL, both its composition (i.e. its sequence when it is a nucleic acid) and its label, is known. Then, by adding the DBLs to the array containing the bioactive agents under conditions which allow the formation of complexes (termed hybridization complexes when the components are nucleic acids) between the DBLs and either the bioactive agents or the IBLs, the location of each DBL can be elucidated. This allows the identification of the location of each capture probe; the random array has been decoded. The DBLs can then be removed, if necessary, and the target sample applied. [0302]
  • In a preferred embodiment, the number of unique labels is less than the number of unique bioactive agents, and thus a sequential series of decoding steps are used. To facilitate the discussion, this embodiment is explained for nucleic acids, although other types of capture probe and DBLs are useful as well. In this embodiment, decoder probes are divided into n sets for decoding. The number of sets corresponds to the number of unique tags. Each decoder probe is labeled in n separate reactions with n distinct tags. All the decoder probes share the same n tags. The decoder probes are pooled so that each pool contains only one of the n tag versions of each decoder, and no two decoder probes have the same sequence of tags across all the pools. The number of pools required for this to be true is determined by the number of decoder probes and the n. Hybridization of each pool to the array generates a signal at every address. The sequential hybridization of each pool in turn will generate a unique, sequence-specific code for each candidate probe. This identifies the candidate probe at each address in the array. For example, if four tags are used, then 4×n sequential hybridizations can ideally distinguish 4[0303] n sequences, although in some cases more steps may be required. After the hybridization of each pool, the hybrids are denatured and the decoder probes removed, so that the probes are rendered single-stranded for the next hybridization (although it is also possible to hybridize limiting amounts of target so that the available probe is not saturated. Sequential hybridizations can be carried out and analyzed by subtracting pre-existing signal from the previous hybridization).
  • An example is illustrative. Assuming an array of 16 probe nucleic acids (numbers 1-16), and four unique tags (four different fluors, for example; labels A-D). Decoder probes 1-16 are made that correspond to the probes on the beads. The first step is to label decoder probes 1-4 with tag A, decoder probes 5-8 with tag B, decoder probes 9-12 with tag C, and decoder probes 13-16 with tag D. The probes are mixed and the pool is contacted with the array containing the beads with the attached candidate probes. The location of each tag (and thus each decoder and candidate probe pair) is then determined. The first set of decoder probes are then removed. A second set is added, but this time, decoder probes 1, 5, 9 and 13 are labeled with tag A, decoder probes 2, 6, 10 and 14 are labeled with tag B, decoder probes 3, 7, 11 and 15 are labeled with tag C, and decoder probes 4, 8, 12 and 16 are labeled with tag D. Thus, those beads that contained tag A in both decoding steps contain candidate probe 1; tag A in the first decoding step and tag B in the second decoding step contain candidate probe 2; tag A in the first decoding step and tag C in the second step contain candidate probe 3; etc. In one embodiment, the decoder probes are labeled in situ; that is, they need not be labeled prior to the decoding reaction. In this embodiment, the incoming decoder probe is shorter than the candidate probe, creating a 5′ “overhang” on the decoding probe. The addition of labeled ddNTPs (each labeled with a unique tag) and a polymerase will allow the addition of the tags in a sequence specific manner, thus creating a sequence-specific pattern of signals. Similarly, other modifications can be done, including ligation, etc. [0304]
  • In addition, since the size of the array will be set by the number of unique decoding binding ligands, it is possible to “reuse” a set of unique DBLs to allow for a greater number of test sites. This may be done in several ways; for example, by using some subpopulations that comprise optical signatures. Similarly, the use of a positional coding scheme within an array; different sub-bundles may reuse the set of DBLs. Similarly, one embodiment utilizes bead size as a coding modality, thus allowing the reuse of the set of unique DBLs for each bead size. Alternatively, sequential partial loading of arrays with beads can also allow the reuse of DBLs. Furthermore, “code sharing” can occur as well. [0305]
  • In a preferred embodiment, the DBLs may be reused by having some subpopulations of beads comprise optical signatures. In a preferred embodiment, the optical signature is generally a mixture of reporter dyes, preferably fluorescent. By varying both the composition of the mixture (i.e. the ratio of one dye to another) and the concentration of the dye (leading to differences in signal intensity), matrices of unique optical signatures may be generated. This may be done by covalently attaching the dyes to the surface of the beads, or alternatively, by entrapping the dye within the bead. The dyes may be chromophores or phosphors but are preferably fluorescent dyes, which due to their strong signals provide a good signal-to-noise ratio for decoding. Suitable dyes for use in the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue™, Texas Red, and others described in the 6th Edition of the Molecular Probes Handbook by Richard P. Haugland, hereby expressly incorporated by reference. [0306]
  • In a preferred embodiment, the encoding can be accomplished in a ratio of at least two dyes, although more encoding dimensions may be added in the size of the beads, for example. In addition, the labels are distinguishable from one another; thus two different labels may comprise different molecules (i.e. two different fluors) or, alternatively, one label at two different concentrations or intensity. [0307]
  • In a preferred embodiment, the dyes are covalently attached to the surface of the beads. This may be done as is generally outlined for the attachment of the capture probe, using functional groups on the surface of the beads. As will be appreciated by those in the art, these attachments are done to minimize the effect on the dye. [0308]
  • In a preferred embodiment, the dyes are non-covalently associated with the beads, generally by entrapping the dyes in the pores of the beads. [0309]
  • Additionally, encoding in the ratios of the two or more dyes, rather than single dye concentrations, is preferred since it provides insensitivity to the intensity of light used to interrogate the reporter dye's signature and detector sensitivity. [0310]
  • In a preferred embodiment, a spatial or positional coding system is done. In this embodiment, there are sub-bundles or subarrays (i.e. portions of the total array) that are utilized. By analogy with the telephone system, each subarray is an “area code”, that can have the same tags (i.e. telephone numbers) of other subarrays, that are separated by virtue of the location of the subarray. Thus, for example, the same unique tags can be reused from bundle to bundle. Thus, the use of 50 unique tags in combination with 100 different subarrays can form an array of 5000 different bioactive agents. In this embodiment, it becomes important to be able to identify one bundle from another; in general, this is done either manually or through the use of marker beads, i.e. beads containing unique tags for each subarray. [0311]
  • In alternative embodiments, additional encoding parameters can be added, such as microsphere size. For example, the use of different size beads may also allow the reuse of sets of DBLs; that is, it is possible to use microspheres of different sizes to expand the encoding dimensions of the microspheres. Optical fiber arrays can be fabricated containing pixels with different fiber diameters or cross-sections; alternatively, two or more fiber optic bundles, each with different cross-sections of the individual fibers, can be added together to form a larger bundle; or, fiber optic bundles with fiber of the same size cross-sections can be used, but just with different sized beads. With different diameters, the largest wells can be filled with the largest microspheres and then moving onto progressively smaller microspheres in the smaller wells until all size wells are then filled. In this manner, the same dye ratio could be used to encode microspheres of different sizes thereby expanding the number of different oligonucleotide sequences or chemical functionalities present in the array. Although outlined for fiber optic substrates, this as well as the other methods outlined herein can be used with other substrates and with other attachment modalities as well. [0312]
  • In a preferred embodiment, the coding and decoding is accomplished by sequential loading of the microspheres into the array. As outlined above for spatial coding, in this embodiment, the optical signatures can be “reused”. In this embodiment, the library of microspheres each comprising a different bioactive agent (or the subpopulations each comprise a different bioactive agent), is divided into a plurality of sublibraries; for example, depending on the size of the desired array and the number of unique tags, 10 sublibraries each comprising roughly 10% of the total library may be made, with each sublibrary comprising roughly the same unique tags. Then, the first sublibrary is added to the fiber optic bundle comprising the wells, and the location of each bioactive agent is determined, generally through the use of DBLs. The second sublibrary is then added, and the location of each bioactive agent is again determined. The signal in this case will comprise the signal from the “first” DBL and the “second” DBL; by comparing the two matrices the location of each bead in each sublibrary can be determined. Similarly, adding the third, fourth, etc. sublibraries sequentially will allow the array to be filled. [0313]
  • In a preferred embodiment, codes can be “shared” in several ways. In a first embodiment, a single code (i.e. IBL/DBL pair) can be assigned to two or more agents if the target analytes different sufficiently in their binding strengths. For example, two nucleic acid probes used in an mRNA quantitation assay can share the same code if the ranges of their hybridization signal intensities do not overlap. This can occur, for example, when one of the target sequences is always present at a much higher concentration than the other. Alternatively, the two target sequences might always be present at a similar concentration, but differ in hybridization efficiency. [0314]
  • Alternatively, a single code can be assigned to multiple agents if the agents are functionally equivalent. For example, if a set of oligonucleotide probes are designed with the common purpose of detecting the presence of a particular gene, then the probes are functionally equivalent, even though they may differ in sequence. Similarly, if classes of analytes are desired, all probes for different members of a class such as kinases or G-protein coupled receptors could share a code. Similarly, an array of this type could be used to detect homologs of known genes. In this embodiment, each gene is represented by a heterologous set of probes, hybridizing to different regions of the gene (and therefore differing in sequence). The set of probes share a common code. If a homolog is present, it might hybridize to some but not all of the probes. The level of homology might be indicated by the fraction of probes hybridizing, as well as the average hybridization intensity. Similarly, multiple antibodies to the same protein could all share the same code. [0315]
  • In a preferred embodiment, several levels of redundancy are built into the arrays of the invention. Building redundancy into an array gives several significant advantages, including the ability to make quantitative estimates of confidence about the data and significant increases in sensitivity. Thus, preferred embodiments utilize array redundancy. As will be appreciated by those in the art, there are at least two types of redundancy that can be built into an array: the use of multiple identical sensor elements (termed herein “sensor redundancy”), and the use of multiple sensor elements directed to the same target analyte, but comprising different chemical functionalities (termed herein “target redundancy”). For example, for the detection of nucleic acids, sensor redundancy utilizes of a plurality of sensor elements such as beads comprising identical binding ligands such as probes. Target redundancy utilizes sensor elements with different probes to the same target: one probe may span the first 25 bases of the target, a second probe may span the second 25 bases of the target, etc. By building in either or both of these types of redundancy into an array, significant benefits are obtained. For example, a variety of statistical mathematical analyses may be done. [0316]
  • In addition, while this is generally described herein for bead arrays, as will be appreciated by those in the art, this techniques can be used for any type of arrays designed to detect target analytes. Furthermore, while these techniques are generally described for nucleic acid systems, these techniques are useful in the detection of other binding ligand/target analyte systems as well. [0317]
  • In a preferred embodiment, sensor redundancy is used. In this embodiment, a plurality of sensor elements, e.g. beads, comprising identical bioactive agents are used. That is, each subpopulation comprises a plurality of beads comprising identical bioactive agents (e.g. binding ligands). By using a number of identical sensor elements for a given array, the optical signal from each sensor element can be combined and any number of statistical analyses run, as outlined below. This can be done for a variety of reasons. For example, in time varying measurements, redundancy can significantly reduce the noise in the system. For non-time based measurements, redundancy can significantly increase the confidence of the data. [0318]
  • In a preferred embodiment, a plurality of identical sensor elements are used. As will be appreciated by those in the art, the number of identical sensor elements will vary with the application and use of the sensor array. In general, anywhere from 2 to thousands may be used, with from 2 to 100 being preferred, 2 to 50 being particularly preferred and from 5 to 20 being especially preferred. In general, preliminary results indicate that roughly 10 beads gives a sufficient advantage, although for some applications, more identical sensor elements can be used. [0319]
  • Once obtained, the optical response signals from a plurality of sensor beads within each bead subpopulation can be manipulated and analyzed in a wide variety of ways, including baseline adjustment, averaging, standard deviation analysis, distribution and cluster analysis, confidence interval analysis, mean testing, etc. [0320]
  • In a preferred embodiment, the first manipulation of the optical response signals is an optional baseline adjustment. In a typical procedure, the standardized optical responses are adjusted to start at a value of 0.0 by subtracting the integer 1.0 from all data points. Doing this allows the baseline-loop data to remain at zero even when summed together and the random response signal noise is canceled out. When the sample is a fluid, the fluid pulse-loop temporal region, however, frequently exhibits a characteristic change in response, either positive, negative or neutral, prior to the sample pulse and often requires a baseline adjustment to overcome noise associated with drift in the first few data points due to charge buildup in the CCD camera. If no drift is present, typically the baseline from the first data point for each bead sensor is subtracted from all the response data for the same bead. If drift is observed, the average baseline from the first ten data points for each bead sensor is substracted from the all the response data for the same bead. By applying this baseline adjustment, when multiple bead responses are added together they can be amplified while the baseline remains at zero. Since all beads respond at the same time to the sample (e.g. the sample pulse), they all see the pulse at the exact same time and there is no registering or adjusting needed for overlaying their responses. In addition, other types of baseline adjustment may be done, depending on the requirements and output of the system used. [0321]
  • Once the baseline has been adjusted, a number of possible statistical analyses may be run to generate known statistical parameters. Analyses based on redundancy are known and generally described in texts such as Freund and Walpole, Mathematical Statistics, Prentice Hall, Inc. New Jersey, 1980, hereby incorporated by reference in its entirety. [0322]
  • In a preferred embodiment, signal summing is done by simply adding the intensity values of all responses at each time point, generating a new temporal response comprised of the sum of all bead responses. These values can be baseline-adjusted or raw. As for all the analyses described herein, signal summing can be performed in real time or during post-data acquisition data reduction and analysis. In one embodiment, signal summing is performed with a commercial spreadsheet program (Excel, Microsoft, Redmond, Wash.) after optical response data is collected. [0323]
  • In a preferred embodiment, cummulative response data is generated by simply adding all data points in successive time intervals. This final column, comprised of the sum of all data points at a particular time interval, may then be compared or plotted with the individual bead responses to determine the extent of signal enhancement or improved signal-to-noise ratios. [0324]
  • In a preferred embodiment, the mean of the subpopulation (i.e. the plurality of identical beads) is determined, using the well known Equation 1: [0325]
  • Equation 1 [0326] μ = x i n Equation 1
    Figure US20020177141A1-20021128-M00001
  • In some embodiments, the subpopulation may be redefined to exclude some beads if necessary (for example for obvious outliers, as discussed below). [0327]
  • In a preferred embodiment, the standard deviation of the subpopulation can be determined, generally using Equation 2 (for the entire subpopulation) and Equation 3 (for less than the entire subpopulation): [0328]
  • Equation 2 [0329] σ = ( x i - μ ) 2 n Equation 2
    Figure US20020177141A1-20021128-M00002
  • Equation 3 [0330] s = ( x i - x _ ) 2 n - 1 Equation 3
    Figure US20020177141A1-20021128-M00003
  • As for the mean, the subpopulation may be redefined to exclude some beads if necessary (for example for obvious outliers, as discussed below). [0331]
  • In a preferred embodiment, statistical analyses are done to evaluate whether a particular data point has statistical validity within a subpopulation by using techniques including, but not limited to, t distribution and cluster analysis. This may be done to statistically discard outliers that may otherwise skew the result and increase the signal-to-noise ratio of any particular experiment. This may be done using Equation 4: [0332]
  • Equation 4 [0333] t = x _ - μ s / n Equation 4
    Figure US20020177141A1-20021128-M00004
  • In a preferred embodiment, the quality of the data is evaluated using confidence intervals, as is known in the art. Confidence intervals can be used to facilitate more comprehensive data processing to measure the statistical validity of a result. [0334]
  • In a preferred embodiment, statistical parameters of a subpopulation of beads are used to do hypothesis testing. One application is tests concerning means, also called mean testing. In this application, statistical evaluation is done to determine whether two subpopulations are different. For example, one sample could be compared with another sample for each subpopulation within an array to determine if the variation is statistically significant. [0335]
  • In addition, mean testing can also be used to differentiate two different assays that share the same code. If the two assays give results that are statistically distinct from each other, then the subpopulations that share a common code can be distinguished from each other on the basis of the assay and the mean test, shown below in Equation 5: [0336]
  • Equation 5 [0337] z = x _ 1 - x _ 2 σ 1 2 n 1 + σ 2 2 n 2 Equation 5
    Figure US20020177141A1-20021128-M00005
  • Furthermore, analyzing the distribution of individual members of a subpopulation of sensor elements may be done. For example, a subpopulation distribution can be evaluated to determine whether the distribution is binomial, Poisson, hypergeometric, etc. [0338]
  • In addition to the sensor redundancy, a preferred embodiment utilizes a plurality of sensor elements that are directed to a single target analyte but yet are not identical. For example, a single target nucleic acid analyte may have two or more sensor elements each comprising a different probe. This adds a level of confidence as non-specific binding interactions can be statistically minimized. When nucleic acid target analytes are to be evaluated, the redundant nucleic acid probes may be overlapping, adjacent, or spatially separated. However, it is preferred that two probes do not compete for a single binding site, so adjacent or separated probes are preferred. Similarly, when proteinaceous target analytes are to be evaluated, preferred embodiments utilize bioactive agent binding agents that bind to different parts of the target. For example, when antibodies (or antibody fragments) are used as bioactive agents for the binding of target proteins, preferred embodiments utilize antibodies to different epitopes. [0339]
  • In this embodiment, a plurality of different sensor elements may be used, with from about 2 to about 20 being preferred, and from about 2 to about 10 being especially preferred, and from 2 to about 5 being particularly preferred, including 2, 3, 4 or 5. However, as above, more may also be used, depending on the application. [0340]
  • As above, any number of statistical analyses may be run on the data from target redundant sensors. [0341]
  • One benefit of the sensor element summing (referred to herein as “bead summing” when beads are used), is the increase in sensitivity that can occur. [0342]
  • Once made, the compositions of the invention find use in a number of applications. [0343]
  • In a preferred embodiment, the probes are used in genetic diagnosis. For example, probes can be made using the techniques disclosed herein to detect target sequences such as the gene for nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which is a gene associated with a variety of cancers, the Apo E4 gene that indicates a greater risk of Alzheimer's disease, allowing for easy presymptomatic screening of patients, mutations in the cystic fibrosis gene, cytochrome p450s or any of the others well known in the art. [0344]
  • In an additional embodiment, viral and bacterial detection is done using the complexes of the invention. In this embodiment, probes are designed to detect target sequences from a variety of bacteria and viruses. For example, current blood-screening techniques rely on the detection of anti-HIV antibodies. The methods disclosed herein allow for direct screening of clinical samples to detect HIV nucleic acid sequences, particularly highly conserved HIV sequences. In addition, this allows direct monitoring of circulating virus within a patient as an improved method of assessing the efficacy of anti-viral therapies. Similarly, viruses associated with leukemia, HTLV-I and HTLV-II, may be detected in this way. Bacterial infections such as tuberculosis, chlamydia and other sexually transmitted diseases, may also be detected. [0345]
  • In a preferred embodiment, the nucleic acids of the invention find use as probes for toxic bacteria in the screening of water and food samples. For example, samples may be treated to lyse the bacteria to release its nucleic acid, and then probes designed to recognize bacterial strains, including, but not limited to, such pathogenic strains as, Salmonella, Campylobacter, [0346] Vibrio cholerae, Leishmania, enterotoxic strains of E. coli, and Legionnaire's disease bacteria. Similarly, bioremediation strategies may be evaluated using the compositions of the invention.
  • In a further embodiment, the probes are used for forensic “DNA fingerprinting” to match crime-scene DNA against samples taken from victims and suspects. [0347]
  • In an additional embodiment, the probes in an array are used for sequencing by hybridization. [0348]
  • The present invention also finds use as a methodology for the detection of mutations or mismatches in target nucleic acid sequences. For example, recent focus has been on the analysis of the relationship between genetic variation and phenotype by making use of polymorphic DNA markers. Previous work utilized short tandem repeats (STRs) as polymorphic positional markers; however, recent focus is on the use of single nucleotide polymorphisms (SNPs), which occur at an average frequency of more than 1 per kilobase in human genomic DNA. Some SNPs, particularly those in and around coding sequences, are likely to be the direct cause of therapeutically relevant phenotypic variants. There are a number of well known polymorphisms that cause clinically important phenotypes; for example, the apoE2/3/4 variants are associated with different relative risk of Alzheimer's and other diseases (see Cordor et al., Science 261(1993). Multiplex PCR amplification of SNP loci with subsequent hybridization to oligonucleotide arrays has been shown to be an accurate and reliable method of simultaneously genotyping at least hundreds of SNPs; see Wang et al., Science, 280:1077 (1998); see also Schafer et al., Nature Biotechnology 16:33-39 (1998). The compositions of the present invention may easily be substituted for the arrays of the prior art. [0349]
  • In a preferred embodiment, a change in optical signal occurs as a result of the binding of a target analyte that is labeled, either directly or indirectly, with a detectable label, preferably an optical label such as a fluorochrome. Thus, for example, when a nucleic acid target analyte is used, it may be either directly labeled with a fluor, or indirectly, for example through the use of a labeled antibody. Similarly, nucleic acids are easily labeled with fluorochromes, as outlined herein. For example during PCR amplification as is known in the art. Alternatively, upon binding of the target sequences, a hybridization indicator may be used as the label. Hybridization indicators preferentially associate with double stranded nucleic acid, usually reversibly. Hybridization indicators include intercalators and minor and/or major groove binding moieties. In a preferred embodiment, intercalators may be used; since intercalation generally only occurs in the presence of double stranded nucleic acid, only in the presence of target hybridization will the label light up. Thus, upon binding of the target analyte to a bioactive agent, there is a new optical signal generated at that site, which then may be detected. [0350]
  • Furthermore, in some embodiments, a change in the optical signature may be the basis of the optical signal. For example, the interaction of some chemical target analytes with some fluorescent dyes on the beads may alter the optical signature, thus generating a different optical signal. [0351]
  • The assays may be run under a variety of experimental conditions, as will be appreciated by those in the art. A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding. Various blocking and washing steps may be utilized as is known in the art. [0352]
  • In a preferred embodiment, the methods of the invention are useful in array quality control. Prior to this invention, no methods have been described that provide a positive test of the performance of every probe on every array. Decoding of the array not only provides this test, it also does so by making use of the data generated during the decoding process itself. Therefore, no additional experimental work is required. The invention requires only a set of data analysis algorithms that can be encoded in software. [0353]
  • The quality control procedure can identify a wide variety of systematic and random problems in an array. For example, random specks of dust or other contaminants might cause some sensors to give an incorrect signal-this can be detected during decoding. The omission of one or more agents from multiple arrays can also be detected. An advantage of this quality control procedure is that it can be implemented immediated prior to the assay itself, and is a true functional test of each individual sensor. Therefore any problems that might occur between array assembly and actual use can be detected. In applications where a very high level of confidence is required, and/or there is a significant chance of sensor failure during the experimental procedure, decoding and quality control can be conducted both before and after the actual sample analysis. [0354]
  • In a preferred embodiment, the arrays can be used to do reagent quality control. In many instances, biological macromolecules are used as reagents and must be quality controlled. For example, large sets of oligonucleotide probes may be provided as reagents. It is typically difficult to perform quality control on large numbers of different biological macromolecules. The approach described here can be used to do this by treating the reagents (formulated as the DBLs) as variable instead of the arrays. [0355]
  • In a preferred embodiment, the methods outlined herein are used in array calibration. For many applications, such as mRNA quantitation, it is desirable to have a signal that is a linear response to the concentration of the target analyte, or, alternatively, if non-linear, to determine a relationship between concentration and signal, so that the concentration of the target analyte can be estimated. Accordingly, the present invention provides methods of creating calibration curves in parallel for multiple beads in an array. The calibration curves can be created under conditions that simulate the complexity of the sample to be analyzed. Each curve can be constructed independently of the others (e.g. for a different range of concentrations), but at the same time as all the other curves for the array. Thus, in this embodiment, the sequential decoding scheme is implemented with different concentrations being used as the code “labels”, rather than different fluorophores. In this way, signal as a response to concentration can be measured for each bead. This calibration can be carried out just prior to array use, so that every probe on every array is individually calibrated as needed. [0356]
  • In a preferred embodiment, the methods of the invention can be used in assay development as well. Thus, for example, the methods allow the identification of good and bad probes; as is understood by those in the art, some probes do not function well because they do not hybridize well, or because they cross-hybridize with more than one sequence. These problems are easily detected during decoding. The ability to rapidly assess probe performance has the potential to greatly reduce the time and expense of assay development. [0357]
  • Similarly, in a preferred embodiment, the methods of the invention are useful in quantitation in assay development. Major challenges of many assays is the ability to detect differences in analyte concentrations between samples, the ability to quantitate these differences, and to measure absolute concentrations of analytes, all in the presence of a complex mixture of related analytes. An example of this problem is the quantitation of a specific mRNA in the presence of total cellular mRNA. One approach that has been developed as a basis of mRNA quantitation makes use of a multiple match and mismatch probe pairs (Lockhart et al., 1996), hereby incorporated by reference in its entirety. While this approach is simple, it requires relatively large numbers of probes. In this approach, a quantitative response to concentration is obtained by averaging the signals from a set of different probes to the gene or sequence of interest. This is necessary because only some probes respond quantitatively, and it is not possible to predict these probes with certainty. In the absence of prior knowledge, only the average response of an appropriately chosen collection of probes is quantitative. However, in the present invention, this can be applied generally to nucleic acid based assays as well as other assays. In essence, the approach is to identify the probes that respond quantitatively in a particular assay, rather than average them with other probes. This is done using the array calibration scheme outlined above, in which concentration-based codes are used. Advantages of this approach include: fewer probes are needed; the accuracy of the measurement is less dependent on the number of probes used; and that the response of the sensors is known with a high level of certainty, since each and every sequence can be tested in an efficient manner. It is important to note that probes that perform well are chosen empirically, which avoids the difficulties and uncertainties of predicting probe performance, particularly in complex sequence mixtures. In contrast, in experiments described to date with ordered arrays, relatively small numbers of sequences are checked by performing quantitative spiking experiments, in which a known mRNA is added to a mixture. [0358]
  • All references cited herein are incorporated by reference in their entirety. [0359]

Claims (26)

We claim:
1. A method for detecting a first target nucleic acid sequence comprising:
a) hybridizing at least a first primer nucleic acid to said first target sequence to form a first hybridization complex;
b) contacting said first hybridization complex with a first enzyme to form a modified first primer nucleic acid;
c) disassociating said first hybridization complex;
d) contacting said modified first primer nucleic acid with an array comprising:
i) a substrate with a surface comprising discrete sites; and
ii) a population of microspheres comprising at least a first subpopulation comprising a first capture probe; such that said first capture probe and the modified primer form an assay complex; wherein said microspheres are distributed on said surface; and
e) detecting the presence of the modified primer nucleic acid.
2. A method according to claim 1 wherein steps a) through c) are repeated prior to step d).
3. A method according to claim 1 further comprising:
a) hybridizing at least a second primer nucleic acid to a second target sequence that is substantially complementary to said first target sequence to form a second hybridization complex;
b) contacting said second hybridization complex with said first enzyme to form a modified second primer nucleic acid;
c) disassociating said second hybridization complex; and
d) forming a second assay complex comprising said modified second primer nucleic acid and a second capture probe on a second subpopulation.
4. A method according to claim 3 wherein steps a) through c) are repeated prior to step d).
5. A method according to claim 2 wherein said first enzyme comprises a DNA polymerase and said modification is an extension of said primer such that the polymerase chain reaction (PCR) occurs.
6. A method according to claim 2 wherein said first enzyme comprises a ligase and said modification comprises a ligation of said first primer which hybridizes to a first domain of said first target sequence to a third primer which hybridizes to a second adjacent domain of said first target sequence, such that the ligase chain reaction (LCR) occurs.
7. A method according to claim 3 wherein said first enzyme comprises a ligase and said modification is a ligation of said second primer which hybridizes to a first domain of said second target sequence to a fourth primer which hybridizes to a second adjacent domain of said second target sequence, such that the ligase chain reaction (LCR) occurs.
8. A method according to claim 2 wherein said first primer comprises a first probe sequence, a first scissile linkage and a second probe sequence, wherein said first enzyme will cleave said first scissile linkage resulting in the separation of said first and said second probe sequences and the disassociation of said first hybridization complex, leaving said first target sequence intact, such that the cycling probe technology (CPT) reaction occurs.
9. A method according to claim 4 wherein said second primer comprises a third probe sequence, a second scissile linkage and a fourth probe sequence, wherein said first enzyme will cleave said second scissile linkage resulting in the separation of said third and said fourth probe sequences and the disassociation of said second hybridization complex, leaving said second target sequence intact, such that the cycling probe technology (CPT) reaction occurs.
10. A method according to claim 2 wherein said first enzyme is a polymerase that extends said first primer and said modified first primer comprises a first newly synthesized strand, and said method further comprises:
a) the addition of a second enzyme comprising a nicking enzyme that nicks said extended first primer leaving said first target sequence intact; and
b) extending from said nick using said polymerase, thereby displacing said first newly synthesized strand and generating a second newly synthesized strand;
such that strand displacement amplification (SDA) occurs.
11. A method according to claim 4 wherein said first enzyme is a polymerase that extends said second primer and said modified first primer comprises a third newly synthesized strand, and said method further comprises:
a) the addition of a second enzyme comprising a nicking enzyme that nicks said extended second primer leaving said second target sequence intact; and
b) extending from said nick using said polymerase, thereby displacing said third newly synthesized strand and generating a fourth newly synthesized strand;
such that strand displacement amplification (SDA) occurs.
12. A method according to claim 2 wherein said first target sequence is a RNA target sequence, said first primer nucleic acid is a DNA primer comprising an RNA polymerase promoter, said first enzyme is a reverse-transcriptase that extends said first primer to form a first newly synthesized DNA strand, and said method further comprises:
a) the addition of a second enzyme comprising an RNA degrading enzyme that degrades said first target sequence;
b) the addition of a third primer that hybridizes to said first newly synthesized DNA strand;
c) the addition of a third enzyme comprising a DNA polymerase that extends said third primer to form a second newly synthesized DNA strand, to form a newly synthesized DNA hybrid;
d) the addition of a fourth enzyme comprising an RNA polymerase that recognizes said RNA polymerase promoter and generates at least one newly synthesized RNA strand from said DNA hybrid;
such that nucleic acid sequence-based amplification (NASBA) occurs.
13. A method according to claim 2 wherein said first primer is an invader primer, said method further comprises hybridizing a signalling primer to said target sequence, said enzyme comprises a structure-specific cleaving enzyme and said modification comprises a cleavage of said signalling primer, such that the invasive cleavage reaction occurs.
14. A method for detecting a target nucleic acid sequence comprising:
a) hybridizing a first primer to a first target sequence to form a first hybridization complex;
b) contacting said first hybridization complex with a first enzyme to extend said first primer to form a first newly synthesized strand and form a nucleic acid hybrid that comprises an RNA polymerase promoter;
c) contacting said hybrid with an RNA polymerase that recognizes said RNA polymerase promoter and generates at least one newly synthesized RNA strand;
d) contacting said newly synthesized RNA strand with an array comprising:
i) a substrate with a surface comprising discrete sites; and
ii) a population of microspheres comprising at least a first subpopulation comprising a first capture probe; such that said first capture probe and the modified primer form an assay complex; wherein said microspheres are distributed on said surface; and
e) detecting the presence of the newly synthesized RNA strand.
15. A method according to claim 14 wherein steps a) through c) are repeated prior to step d).
16. A method according to claim 14 wherein said target nucleic acid sequence is a RNA sequence, and prior to step a), said method comprises:
f) hybridizing a second primer comprising an RNA polymerase promoter sequence to said RNA sequence to form a second hybridization complex;
g) contacting said second hybridization complex with a second enzyme to extend said second primer to form a second newly synthesized strand and form a nucleic acid hybrid; and
h) degrading said RNA sequence to leave said second newly synthesized strand as said first target sequence.
17. A method according to claim 16 wherein said degrading is done by the addition of an RNA degrading enzyme.
18. A method according to claim 16 wherein said degrading is done by RNA degrading activity of said reverse transcriptase.
19. A method according to claim 14 wherein said target nucleic acid sequence is a DNA sequence, and prior to step a), said method comprises:
f) hybridizing a second primer comprising an RNA polymerase promoter sequence to said DNA sequence to form a second hybridization complex;
g) contacting said second hybridization complex with a second enzyme to extend said second primer to form a second newly synthesized strand and form a nucleic acid hybrid; and
h) denaturing said nucleic acid hybrid such that said second newly synthesized strand is said first target sequence.
20. A method according to claim 1 wherein said first primer nucleic acid hybridizes at its 5′ end to said target nucleic acid sequence and at its 3′ end to a sequence immediately adjacent to said 5′ end, wherein said first enzyme comprises a ligase and said modification comprises ligation of said 5′ end with said 3′ end to form a circular probe, wherein said second enzyme is a polymerase and said amplification is an amplification of said circular probe such that rolling circle amplification occurs.
21. A kit for the detection of a first target nucleic acid sequence comprising:
a) at least a first nucleic acid primer substantially complementary to at least a first domain of said target sequence;
b) at least a first enzyme that will modify said first nucleic acid primer; and
c) an array comprising:
i) a substrate with a surface comprising discrete sites; and
ii) a population of microspheres comprising at least a first and a second subpopulation, wherein each subpopulation comprises a bioactive agent; wherein said microspheres are distributed on said surface.
22. A kit according to claim 21 for the detection of a PCR reaction wherein said first enzyme is a thermostable DNA polymerase.
23. A kit according to claim 21 for the detection of a LCR reaction wherein said first enzyme is a ligase and said kit comprises a first nucleic acid primer substantially complementary to a first domain of said first target sequence and a third nucleic acid primer substantially complementary to a second adjacent domain of said first target sequence.
24. A kit according to claim 21 for the detection of a strand displacement amplification (SDA) reaction wherein said first enzyme is a polymerase and said kit further comprises a nicking enzyme.
25. A kit according to claim 21 for the detection of a NASBA reaction wherein said first enzyme is a reverse transcriptase, and said kit comprises a second enzyme comprising an RNA degrading enzyme, a third primer, a third enzyme comprising a DNA polymerase and a fourth enzyme comprising an RNA polymerase.
26. A kit according to claim 21 for the detection of an invasive cleavage reaction wherein said first enzyme is a structure-specific cleaving enzyme, and said kit comprises a signalling primer.
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Cited By (50)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020164628A1 (en) * 2001-03-09 2002-11-07 Nurith Kurn Methods and compositions for amplification of RNA sequences
US20030087251A1 (en) * 2001-03-09 2003-05-08 Nurith Kurn Methods and compositions for amplification of RNA sequences
US20040171054A1 (en) * 1994-06-08 2004-09-02 Affymetrix, Inc. Bioarray chip reaction apparatus and its manufacture
US20050019793A1 (en) * 2003-04-14 2005-01-27 Nurith Kurn Global amplification using a randomly primed composite primer
US20050026200A1 (en) * 2003-07-28 2005-02-03 Holden David P. Method for error detection and increased confidence of sample decoding
US20050048667A1 (en) * 2003-08-29 2005-03-03 Brett Ellman Method of forming and using solid-phase support
US20050089851A1 (en) * 2001-01-25 2005-04-28 Petr Pancoska Polynucleotides for use as tags and tag complements, manufacture and use thereof
US20050181403A1 (en) * 1995-06-07 2005-08-18 Affymetrix, Inc. Methods for making a device for concurrently processing multiple biological chip assays
WO2005117514A3 (en) * 2004-06-01 2007-05-18 Tm Bioscience Corp Method of detecting cystic fibrosis associated mutations
US20080161194A1 (en) * 2004-09-17 2008-07-03 Stephen Turner Arrays of optical confinements and uses thereof
US20090062129A1 (en) * 2006-04-19 2009-03-05 Agencourt Personal Genomics, Inc. Reagents, methods, and libraries for gel-free bead-based sequencing
US20090181390A1 (en) * 2008-01-11 2009-07-16 Signosis, Inc. A California Corporation High throughput detection of micrornas and use for disease diagnosis
US20090181860A1 (en) * 2005-02-01 2009-07-16 Applied Biosystems Inc. Reagents, methods, and libraries for bead-based sequencing
US20090191553A1 (en) * 2007-10-01 2009-07-30 Applied Biosystems Inc. Chase Ligation Sequencing
US20090286286A1 (en) * 2007-11-06 2009-11-19 Ambergen , Inc. Methods for controlling amplification
US7645868B2 (en) 2001-01-25 2010-01-12 Luminex Molecular Diagnostics, Inc. Families of non-cross-hybridizing polynucleotides for use as tags and tag complements, manufacture and use thereof
US20100081576A1 (en) * 2008-10-01 2010-04-01 Ach Robert A Method for genome analysis
US7771934B2 (en) 2000-12-13 2010-08-10 Nugen Technologies, Inc. Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof
US7846666B2 (en) 2008-03-21 2010-12-07 Nugen Technologies, Inc. Methods of RNA amplification in the presence of DNA
US7846733B2 (en) 2000-06-26 2010-12-07 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
US7901897B2 (en) 1998-12-28 2011-03-08 Illumina, Inc. Methods of making arrays
US7939258B2 (en) 2005-09-07 2011-05-10 Nugen Technologies, Inc. Nucleic acid amplification procedure using RNA and DNA composite primers
WO2011057061A1 (en) 2009-11-06 2011-05-12 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US7955794B2 (en) 2000-09-21 2011-06-07 Illumina, Inc. Multiplex nucleic acid reactions
EP2366801A1 (en) 2006-06-14 2011-09-21 Verinata Health, Inc Methods for the diagnosis of fetal abnormalities
US8034568B2 (en) 2008-02-12 2011-10-11 Nugen Technologies, Inc. Isothermal nucleic acid amplification methods and compositions
US8076063B2 (en) 2000-02-07 2011-12-13 Illumina, Inc. Multiplexed methylation detection methods
US8288103B2 (en) 2000-02-07 2012-10-16 Illumina, Inc. Multiplex nucleic acid reactions
WO2013043922A1 (en) 2011-09-22 2013-03-28 ImmuMetrix, LLC Compositions and methods for analyzing heterogeneous samples
EP2589668A1 (en) 2006-06-14 2013-05-08 Verinata Health, Inc Rare cell analysis using sample splitting and DNA tags
US8460865B2 (en) 1998-06-24 2013-06-11 Illumina, Inc. Multiplex decoding of array sensors with microspheres
WO2013085710A2 (en) 2011-12-09 2013-06-13 Illumina, Inc. Expanded radix for polymeric tags
US8796186B2 (en) 2005-04-06 2014-08-05 Affymetrix, Inc. System and method for processing large number of biological microarrays
EP2824180A4 (en) * 2012-03-05 2016-03-02 Ngk Insulators Ltd Method for detecting target nucleic acid
US9714937B2 (en) 2009-10-13 2017-07-25 Nanostring Technologies, Inc. Protein detection via nanoreporters
US9714446B2 (en) 2010-02-11 2017-07-25 Nanostring Technologies, Inc. Compositions and methods for the detection of small RNAs
US9758834B2 (en) 2011-03-28 2017-09-12 Nanostring Technologies, Inc. Compositions and methods for diagnosing cancer
US9856519B2 (en) 2008-08-14 2018-01-02 Nanostring Technologies, Inc. Stable nanoreporters
US9890419B2 (en) 2005-12-23 2018-02-13 Nanostring Technologies, Inc. Nanoreporters and methods of manufacturing and use thereof
US9920380B2 (en) 2001-07-03 2018-03-20 The Institute For Systems Biology Methods for detection and quantification of analytes in complex mixtures
WO2018107054A1 (en) * 2016-12-09 2018-06-14 Ultivue, Inc. Improved methods for multiplex imaging using labeled nucleic acid imaging agents
EP3424598A1 (en) 2006-06-14 2019-01-09 Verinata Health, Inc Rare cell analysis using sample splitting and dna tags
US10415080B2 (en) 2016-11-21 2019-09-17 Nanostring Technologies, Inc. Chemical compositions and methods of using same
WO2019229792A1 (en) * 2018-05-28 2019-12-05 株式会社ニコン Method for detecting target nucleic acid fragment
US10501777B2 (en) 2015-07-17 2019-12-10 Nanostring Technologies, Inc. Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue
US10640816B2 (en) 2015-07-17 2020-05-05 Nanostring Technologies, Inc. Simultaneous quantification of gene expression in a user-defined region of a cross-sectioned tissue
US10722858B2 (en) 2013-03-15 2020-07-28 Lineage Biosciences, Inc. Methods and compositions for tagging and analyzing samples
US11377689B2 (en) 2018-02-12 2022-07-05 Nanostring Technologies, Inc. Chemical compositions and uses thereof
US11549139B2 (en) 2018-05-14 2023-01-10 Nanostring Technologies, Inc. Chemical compositions and methods of using same
US11705217B2 (en) 2008-03-28 2023-07-18 Pacific Biosciences Of California, Inc. Sequencing using concatemers of copies of sense and antisense strands

Families Citing this family (454)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5846717A (en) * 1996-01-24 1998-12-08 Third Wave Technologies, Inc. Detection of nucleic acid sequences by invader-directed cleavage
DE69527585T2 (en) * 1994-06-08 2003-04-03 Affymetrix Inc Method and device for packaging chips
US20070269799A9 (en) * 1994-06-22 2007-11-22 Zhang David Y Nucleic acid amplification methods
GB9507238D0 (en) * 1995-04-07 1995-05-31 Isis Innovation Detecting dna sequence variations
US5854033A (en) * 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
US7432048B2 (en) * 1996-11-29 2008-10-07 Third Wave Technologies, Inc. Reactions on a solid surface
DE69737883T2 (en) 1996-04-25 2008-03-06 Bioarray Solutions Ltd. LIGHT-REGULATED, ELECTROKINETIC COMPOSITION OF PARTICLES TO SURFACES
US7622294B2 (en) * 1997-03-14 2009-11-24 Trustees Of Tufts College Methods for detecting target analytes and enzymatic reactions
US20030027126A1 (en) 1997-03-14 2003-02-06 Walt David R. Methods for detecting target analytes and enzymatic reactions
US6908770B1 (en) * 1998-07-16 2005-06-21 Board Of Regents, The University Of Texas System Fluid based analysis of multiple analytes by a sensor array
AU1219800A (en) * 1998-10-20 2000-05-08 Ljl Biosystems, Inc. Improvements in luminescence assays
MXPA01005267A (en) * 1998-11-27 2002-04-24 Synaptics Uk Ltd Position sensor.
US20060275782A1 (en) * 1999-04-20 2006-12-07 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
US7211390B2 (en) * 1999-09-16 2007-05-01 454 Life Sciences Corporation Method of sequencing a nucleic acid
US7244559B2 (en) * 1999-09-16 2007-07-17 454 Life Sciences Corporation Method of sequencing a nucleic acid
CA2389769A1 (en) * 1999-11-02 2001-05-10 Celine Hu Molecular microarrays and methods for production and use thereof
WO2001055704A1 (en) * 2000-01-31 2001-08-02 Board Of Regents, The University Of Texas System System for transferring fluid samples through a sensor array
US20050214825A1 (en) * 2000-02-07 2005-09-29 John Stuelpnagel Multiplex sample analysis on universal arrays
US7611869B2 (en) * 2000-02-07 2009-11-03 Illumina, Inc. Multiplexed methylation detection methods
US6913884B2 (en) * 2001-08-16 2005-07-05 Illumina, Inc. Compositions and methods for repetitive use of genomic DNA
US7361488B2 (en) * 2000-02-07 2008-04-22 Illumina, Inc. Nucleic acid detection methods using universal priming
EP1990428B1 (en) * 2000-02-07 2010-12-22 Illumina, Inc. Nucleic acid detection methods using universal priming
US6770441B2 (en) * 2000-02-10 2004-08-03 Illumina, Inc. Array compositions and methods of making same
DE60136335D1 (en) 2000-02-16 2008-12-11 Illumina Inc PARALLEL GENOTYPING OF SEVERAL PATIENT SAMPLES
EP1320628A2 (en) * 2000-04-05 2003-06-25 Molecular Staging Inc. Process for allele discrimation utilizing primer extension
AU2001270504A1 (en) * 2000-05-04 2001-11-12 Syngenta Participations Ag Novel assay for nucleic acid analysis
US6917726B2 (en) * 2001-09-27 2005-07-12 Cornell Research Foundation, Inc. Zero-mode clad waveguides for performing spectroscopy with confined effective observation volumes
EP1164201A1 (en) * 2000-06-14 2001-12-19 Facultés Universitaires Notre-Dame de la Paix Reverse detection for identification and/or quantification of nucleotide target sequences on biochips
US9709559B2 (en) 2000-06-21 2017-07-18 Bioarray Solutions, Ltd. Multianalyte molecular analysis using application-specific random particle arrays
EP1311839B1 (en) 2000-06-21 2006-03-01 Bioarray Solutions Ltd Multianalyte molecular analysis using application-specific random particle arrays
US6323009B1 (en) * 2000-06-28 2001-11-27 Molecular Staging, Inc. Multiply-primed amplification of nucleic acid sequences
US7807447B1 (en) * 2000-08-25 2010-10-05 Merck Sharp & Dohme Corp. Compositions and methods for exon profiling
US6713257B2 (en) * 2000-08-25 2004-03-30 Rosetta Inpharmatics Llc Gene discovery using microarrays
CH699253B1 (en) * 2000-09-18 2010-02-15 Eidgenoessische Forschungsanst A method of characterizing and / or identification of genomes.
US6806051B2 (en) * 2000-09-25 2004-10-19 Picoliter Inc. Arrays of partially nonhybridizing oligonucleotides and preparation thereof using focused acoustic energy
US6803196B1 (en) 2000-10-13 2004-10-12 Affymetrix, Inc. Methods and compositions for detecting signals in binding assays using microparticles
JP4287652B2 (en) * 2000-10-24 2009-07-01 ザ・ボード・オブ・トラスティーズ・オブ・ザ・レランド・スタンフォード・ジュニア・ユニバーシティ Characterization of genomic DNA by direct multiple processing
EP1203945B1 (en) * 2000-10-26 2006-12-20 Agilent Technologies, Inc. (a Delaware corporation) Microarray
US20040018491A1 (en) * 2000-10-26 2004-01-29 Kevin Gunderson Detection of nucleic acid reactions on bead arrays
CA2428732C (en) * 2000-11-15 2018-07-31 Minerva Biotechnologies Corporation Methods and kit for simultaneous screening of interactions between a plurality of chemical or biological species and uses thereof
US20020197622A1 (en) * 2001-01-31 2002-12-26 Mcdevitt John T. Method and apparatus for the confinement of materials in a micromachined chemical sensor array
CA2442939A1 (en) * 2001-04-02 2002-10-17 Point-2-Point Genomics Ltd. Polynucleotide analysis using combinatorial pcr
US7138506B2 (en) * 2001-05-09 2006-11-21 Genetic Id, Na, Inc. Universal microarray system
WO2004059289A2 (en) * 2001-05-22 2004-07-15 Epicentre Technologies Target-dependent transcription using deletion mutants of n4 rna polymerase
PT2070939E (en) 2001-05-25 2014-06-09 Univ Duke Modulators of pharmacological agents
EP1395805A4 (en) * 2001-06-11 2005-03-09 Illumina Inc Multiplexed detection methods
AU2002305478A1 (en) * 2001-06-20 2003-01-08 Molecular Staging, Inc. Conjugates of reduced antibodies and biomolecules
US7262063B2 (en) 2001-06-21 2007-08-28 Bio Array Solutions, Ltd. Directed assembly of functional heterostructures
US20030170695A1 (en) * 2001-06-29 2003-09-11 Liang Shi Enzymatic ligation-based identification of nucleotide sequences
US20030082584A1 (en) * 2001-06-29 2003-05-01 Liang Shi Enzymatic ligation-based identification of transcript expression
CA2453207A1 (en) * 2001-07-06 2003-01-16 454 Corporation Method for isolation of independent, parallel chemical micro-reactions using a porous filter
CA2454319A1 (en) 2001-07-26 2003-03-27 Stratagene Multi-site mutagenesis
US20030040129A1 (en) * 2001-08-20 2003-02-27 Shah Haresh P. Binding assays using magnetically immobilized arrays
US20030054396A1 (en) * 2001-09-07 2003-03-20 Weiner Michael P. Enzymatic light amplification
ES2661167T3 (en) 2001-10-15 2018-03-27 Bioarray Solutions Ltd. Multiplexed analysis of polymorphic loci by simultaneous consultation and enzyme-mediated detection
US6902921B2 (en) 2001-10-30 2005-06-07 454 Corporation Sulfurylase-luciferase fusion proteins and thermostable sulfurylase
US6956114B2 (en) 2001-10-30 2005-10-18 '454 Corporation Sulfurylase-luciferase fusion proteins and thermostable sulfurylase
US20050124022A1 (en) * 2001-10-30 2005-06-09 Maithreyan Srinivasan Novel sulfurylase-luciferase fusion proteins and thermostable sulfurylase
AU2002366179A1 (en) * 2001-11-20 2003-06-10 The Regents Of The University Of California Early leukemia diagnostics using microsphere arrays
EP1448800A4 (en) * 2001-11-21 2007-05-16 Applera Corp Digital assay
AU2002359645A1 (en) * 2001-12-07 2003-07-09 University Of Massachusetts Targeted genetic risk-stratification using microarrays
US8765484B2 (en) * 2002-02-07 2014-07-01 The Regents Of The University Of California Optically encoded particles
US7553619B2 (en) * 2002-02-08 2009-06-30 Qiagen Gmbh Detection method using dissociated rolling circle amplification
AU2003228711C1 (en) 2002-04-26 2010-01-07 Board Of Regents, The University Of Texas System Method and system for the detection of cardiac risk factors
US9388459B2 (en) * 2002-06-17 2016-07-12 Affymetrix, Inc. Methods for genotyping
US7108976B2 (en) * 2002-06-17 2006-09-19 Affymetrix, Inc. Complexity management of genomic DNA by locus specific amplification
AU2003269908A1 (en) * 2002-07-15 2004-02-02 Illumina, Inc. Multiplex nucleic acid reactions
JP2004053417A (en) * 2002-07-19 2004-02-19 National Institute Of Advanced Industrial & Technology Method for analyzing molecules using micro channel
US7183104B1 (en) 2002-08-23 2007-02-27 Duane Morris Llp Separator and particle detection system
US20040137468A1 (en) * 2002-08-30 2004-07-15 Brian Warner Solid phase based nucleic acid assays combining high affinity and high specificity
US7157228B2 (en) * 2002-09-09 2007-01-02 Bioarray Solutions Ltd. Genetic analysis and authentication
US20040259105A1 (en) * 2002-10-03 2004-12-23 Jian-Bing Fan Multiplex nucleic acid analysis using archived or fixed samples
US7459273B2 (en) * 2002-10-04 2008-12-02 Affymetrix, Inc. Methods for genotyping selected polymorphism
US20040086867A1 (en) * 2002-10-30 2004-05-06 Jian Han Method for detecting nucleic acid
AU2003298655A1 (en) 2002-11-15 2004-06-15 Bioarray Solutions, Ltd. Analysis, secure access to, and transmission of array images
US20040121338A1 (en) * 2002-12-19 2004-06-24 Alsmadi Osama A. Real-time detection of rolling circle amplification products
US9487823B2 (en) * 2002-12-20 2016-11-08 Qiagen Gmbh Nucleic acid amplification
US20040175737A1 (en) * 2002-12-23 2004-09-09 Wyeth Assay for RNase H activity
US7887752B2 (en) * 2003-01-21 2011-02-15 Illumina, Inc. Chemical reaction monitor
US7323305B2 (en) * 2003-01-29 2008-01-29 454 Life Sciences Corporation Methods of amplifying and sequencing nucleic acids
US7575865B2 (en) * 2003-01-29 2009-08-18 454 Life Sciences Corporation Methods of amplifying and sequencing nucleic acids
US20040175843A1 (en) * 2003-03-04 2004-09-09 Roitman Daniel B. Near-field and far-field encoding and shaping of microbeads for bioassays
US20040175842A1 (en) * 2003-03-04 2004-09-09 Roitman Daniel B. Near-field and far-field encoding of microbeads for bioassays
US8043834B2 (en) * 2003-03-31 2011-10-25 Qiagen Gmbh Universal reagents for rolling circle amplification and methods of use
US7960131B2 (en) * 2003-04-21 2011-06-14 Wisconsin Alumni Research Foundation Functional genomic pore assay for mixed cell populations
US20040248103A1 (en) * 2003-06-04 2004-12-09 Feaver William John Proximity-mediated rolling circle amplification
US20040259100A1 (en) 2003-06-20 2004-12-23 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
EP1636337A4 (en) 2003-06-20 2007-07-04 Illumina Inc Methods and compositions for whole genome amplification and genotyping
US20050181394A1 (en) * 2003-06-20 2005-08-18 Illumina, Inc. Methods and compositions for whole genome amplification and genotyping
US8048627B2 (en) * 2003-07-05 2011-11-01 The Johns Hopkins University Method and compositions for detection and enumeration of genetic variations
US8114978B2 (en) * 2003-08-05 2012-02-14 Affymetrix, Inc. Methods for genotyping selected polymorphism
WO2005074417A2 (en) * 2003-09-03 2005-08-18 Salk Institute For Biological Studies Multiple antigen detection assays and reagents
US7365179B2 (en) * 2003-09-09 2008-04-29 Compass Genetics, Llc Multiplexed analytical platform
WO2005029705A2 (en) 2003-09-18 2005-03-31 Bioarray Solutions, Ltd. Number coding for identification of subtypes of coded types of solid phase carriers
US20050100939A1 (en) * 2003-09-18 2005-05-12 Eugeni Namsaraev System and methods for enhancing signal-to-noise ratios of microarray-based measurements
WO2005031305A2 (en) 2003-09-22 2005-04-07 Bioarray Solutions, Ltd. Surface immobilized polyelectrolyte with multiple functional groups capable of covalently bonding to biomolecules
US20050250117A1 (en) * 2003-10-07 2005-11-10 Xing Su Isolation of single polymeric molecules
EP1692298A4 (en) 2003-10-28 2008-08-13 Bioarray Solutions Ltd Optimization of gene expression analysis using immobilized capture probes
CN1882703B (en) 2003-10-29 2011-07-06 佰尔瑞溶液有限公司 Multiplexed nucleic acid analysis by fragmentation of double-stranded DNA
EP1695082A2 (en) * 2003-12-11 2006-08-30 Board of Regents, The University of Texas System Method and system for the analysis of saliva using a sensor array
CN1918582A (en) * 2003-12-22 2007-02-21 加利福尼亚大学董事会 Optically encoded particles with grey scale spectra
CN1918304B (en) * 2003-12-22 2011-08-10 加利福尼亚大学董事会 Optically encoded particles, system and high-throughput screening
US20050136414A1 (en) * 2003-12-23 2005-06-23 Kevin Gunderson Methods and compositions for making locus-specific arrays
AU2005214329A1 (en) 2004-02-12 2005-09-01 Population Genetics Technologies Ltd Genetic analysis by sequence-specific sorting
WO2005082110A2 (en) * 2004-02-26 2005-09-09 Illumina Inc. Haplotype markers for diagnosing susceptibility to immunological conditions
US8105849B2 (en) * 2004-02-27 2012-01-31 Board Of Regents, The University Of Texas System Integration of fluids and reagents into self-contained cartridges containing sensor elements
US8101431B2 (en) * 2004-02-27 2012-01-24 Board Of Regents, The University Of Texas System Integration of fluids and reagents into self-contained cartridges containing sensor elements and reagent delivery systems
WO2005086679A2 (en) * 2004-03-05 2005-09-22 Medical College Of Ohio Methods and compositions for assessing nucleic acids and alleles
WO2005090316A1 (en) * 2004-03-12 2005-09-29 Wyeth HYDANTOINS HAVING RNase MODULATORY ACTIVITY
US7476502B2 (en) * 2004-04-12 2009-01-13 Willey James C Methods and compositions for assaying analytes
US8216810B2 (en) * 2004-04-27 2012-07-10 The Trustees Of Columbia University In The City Of New York Multiplex systems, methods, and kits for detecting and identifying nucleic acids
US7407816B2 (en) 2004-05-07 2008-08-05 Gentius, Inc Isoelectric particles and uses thereof
WO2005111242A2 (en) * 2004-05-10 2005-11-24 Parallele Bioscience, Inc. Digital profiling of polynucleotide populations
EP1756307A1 (en) * 2004-05-20 2007-02-28 Trillion Genomics Limited Use of mass labelled probes to detect target nucleic acids using mass spectrometry
US20050260607A1 (en) * 2004-05-21 2005-11-24 Kao Cheng S Single nucleotide polymorphism genotyping
US7338763B2 (en) * 2004-06-02 2008-03-04 Eppendorf Array Technologies S.A. Method and kit for the detection and/or quantification of homologous nucleotide sequences on arrays
US20070249024A1 (en) * 2004-06-03 2007-10-25 Kanae Muriso Novel Dna Synthesis Technology with 3'-Beaded Oligo Dna and Dna Polymerase
US8536661B1 (en) 2004-06-25 2013-09-17 University Of Hawaii Biosensor chip sensor protection methods
US7702466B1 (en) 2004-06-29 2010-04-20 Illumina, Inc. Systems and methods for selection of nucleic acid sequence probes
US20060019258A1 (en) * 2004-07-20 2006-01-26 Illumina, Inc. Methods and compositions for detection of small interfering RNA and micro-RNA
US20060019304A1 (en) * 2004-07-26 2006-01-26 Paul Hardenbol Simultaneous analysis of multiple genomes
US7848889B2 (en) 2004-08-02 2010-12-07 Bioarray Solutions, Ltd. Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification
WO2007008246A2 (en) 2004-11-12 2007-01-18 The Board Of Trustees Of The Leland Stanford Junior University Charge perturbation detection system for dna and other molecules
US7647186B2 (en) * 2004-12-07 2010-01-12 Illumina, Inc. Oligonucleotide ordering system
WO2006086668A2 (en) * 2005-02-09 2006-08-17 Epicentre Technologies Compositions and methods employing 5'-phosphate-dependent nucleic acid exonucleases
US7407757B2 (en) * 2005-02-10 2008-08-05 Population Genetics Technologies Genetic analysis by sequence-specific sorting
US20070108465A1 (en) * 2005-03-10 2007-05-17 The Regents Of The University Of California Porous microstructure multi layer spectroscopy and biosensing
US7544793B2 (en) * 2005-03-10 2009-06-09 Xialoian Gao Making nucleic acid sequences in parallel and use
US20060211030A1 (en) * 2005-03-16 2006-09-21 Sydney Brenner Methods and compositions for assay readouts on multiple analytical platforms
US8309303B2 (en) * 2005-04-01 2012-11-13 Qiagen Gmbh Reverse transcription and amplification of RNA with simultaneous degradation of DNA
US7452671B2 (en) * 2005-04-29 2008-11-18 Affymetrix, Inc. Methods for genotyping with selective adaptor ligation
US8377398B2 (en) 2005-05-31 2013-02-19 The Board Of Regents Of The University Of Texas System Methods and compositions related to determination and use of white blood cell counts
US8486629B2 (en) 2005-06-01 2013-07-16 Bioarray Solutions, Ltd. Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation
US7368242B2 (en) * 2005-06-14 2008-05-06 Affymetrix, Inc. Method and kits for multiplex hybridization assays
US7709197B2 (en) 2005-06-15 2010-05-04 Callida Genomics, Inc. Nucleic acid analysis by random mixtures of non-overlapping fragments
DE102005029810B4 (en) * 2005-06-27 2008-11-13 Siemens Ag Method for detecting nucleotide sequences, use of the method and test set
EP1762627A1 (en) 2005-09-09 2007-03-14 Qiagen GmbH Method for the activation of a nucleic acid for performing a polymerase reaction
US9360526B2 (en) 2005-10-24 2016-06-07 The Johns Hopkins University Methods for beaming
US7329860B2 (en) 2005-11-23 2008-02-12 Illumina, Inc. Confocal imaging methods and apparatus
JP2007183257A (en) * 2005-12-09 2007-07-19 Canon Inc Method for acquiring reaction data from probe fixing carrier
US7803751B2 (en) * 2005-12-09 2010-09-28 Illumina, Inc. Compositions and methods for detecting phosphomonoester
US11306351B2 (en) 2005-12-21 2022-04-19 Affymetrix, Inc. Methods for genotyping
US7537897B2 (en) * 2006-01-23 2009-05-26 Population Genetics Technologies, Ltd. Molecular counting
GB0601302D0 (en) * 2006-01-23 2006-03-01 Semikhodskii Andrei Diagnostic methods and apparatus
US8673567B2 (en) * 2006-03-08 2014-03-18 Atila Biosystems, Inc. Method and kit for nucleic acid sequence detection
US7914988B1 (en) 2006-03-31 2011-03-29 Illumina, Inc. Gene expression profiles to predict relapse of prostate cancer
DE102006020885A1 (en) * 2006-05-05 2007-11-08 Qiagen Gmbh Inserting a tag sequence into a nucleic acid comprises using an anchor oligonucleotide comprising a hybridizing anchor sequence and a nonhybridizing tag-template sequence
US20080003667A1 (en) * 2006-05-19 2008-01-03 Affymetrix, Inc. Consumable elements for use with fluid processing and detection systems
EP2527501A1 (en) 2006-11-15 2012-11-28 The University Of British Columbia Polymorphisms predictive of anthracycline-induced cardiotoxicity
US20080242560A1 (en) * 2006-11-21 2008-10-02 Gunderson Kevin L Methods for generating amplified nucleic acid arrays
KR100823684B1 (en) * 2006-12-06 2008-04-21 한국전자통신연구원 Method for detecting a biological target material using barcode dna
US8262900B2 (en) 2006-12-14 2012-09-11 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
EP2639579B1 (en) 2006-12-14 2016-11-16 Life Technologies Corporation Apparatus for measuring analytes using large scale FET arrays
US11339430B2 (en) 2007-07-10 2022-05-24 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
US8349167B2 (en) 2006-12-14 2013-01-08 Life Technologies Corporation Methods and apparatus for detecting molecular interactions using FET arrays
US20080241831A1 (en) * 2007-03-28 2008-10-02 Jian-Bing Fan Methods for detecting small RNA species
US20080274458A1 (en) * 2007-05-01 2008-11-06 Latham Gary J Nucleic acid quantitation methods
US7769548B2 (en) * 2007-05-10 2010-08-03 Illumina, Inc. Microarray analytical data stitching system and method
US20080293589A1 (en) * 2007-05-24 2008-11-27 Affymetrix, Inc. Multiplex locus specific amplification
CN101720359A (en) * 2007-06-01 2010-06-02 454生命科学公司 System and meth0d for identification of individual samples from a multiplex mixture
US20110105366A1 (en) 2007-06-18 2011-05-05 Illumina, Inc. Microfabrication methods for the optimal patterning of substrates
GB0713183D0 (en) * 2007-07-06 2007-08-15 King S College London Method
FR2920598B1 (en) * 2007-09-03 2010-08-27 Eurocopter France ELECTRICAL CONNECTION OF A FIRST AND A SECOND ELECTRICALLY ISOLATED ELECTRICALLY INSULATED METALLIC PIECES
US9388457B2 (en) * 2007-09-14 2016-07-12 Affymetrix, Inc. Locus specific amplification using array probes
US8202691B2 (en) * 2008-01-25 2012-06-19 Illumina, Inc. Uniform fragmentation of DNA using binding proteins
US9074244B2 (en) 2008-03-11 2015-07-07 Affymetrix, Inc. Array-based translocation and rearrangement assays
US8039817B2 (en) 2008-05-05 2011-10-18 Illumina, Inc. Compensator for multiple surface imaging
US20110105356A1 (en) * 2008-05-07 2011-05-05 Derosier Chad F Compositions and methods for providing substances to and from an array
JP2009268665A (en) * 2008-05-07 2009-11-19 Canon Inc Inhalation device
CN101586150B (en) * 2008-05-23 2016-09-28 陕西佰美基因股份有限公司 Detection probe, general oligonucleotide chip and nucleic acid detection method and application thereof
EP2982437B1 (en) 2008-06-25 2017-12-06 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale fet arrays
WO2010003132A1 (en) 2008-07-02 2010-01-07 Illumina Cambridge Ltd. Using populations of beads for the fabrication of arrays on surfaces
US8383345B2 (en) 2008-09-12 2013-02-26 University Of Washington Sequence tag directed subassembly of short sequencing reads into long sequencing reads
US8945912B2 (en) 2008-09-29 2015-02-03 The Board Of Trustees Of The University Of Illinois DNA sequencing and amplification systems using nanoscale field effect sensor arrays
US20100087325A1 (en) * 2008-10-07 2010-04-08 Illumina, Inc. Biological sample temperature control system and method
US20100137143A1 (en) 2008-10-22 2010-06-03 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
US20100301398A1 (en) 2009-05-29 2010-12-02 Ion Torrent Systems Incorporated Methods and apparatus for measuring analytes
WO2010048337A2 (en) 2008-10-22 2010-04-29 Illumina, Inc. Preservation of information related to genomic dna methylation
WO2010068702A2 (en) * 2008-12-10 2010-06-17 Illumina, Inc. Methods and compositions for hybridizing nucleic acids
WO2010099230A2 (en) 2009-02-25 2010-09-02 Illumina, Inc. Separation of pyrophosphate release and pyrophosphate detection
CN102369297B (en) * 2009-03-31 2016-07-06 凸版印刷株式会社 Identify genotypic method
US8776573B2 (en) 2009-05-29 2014-07-15 Life Technologies Corporation Methods and apparatus for measuring analytes
US8673627B2 (en) 2009-05-29 2014-03-18 Life Technologies Corporation Apparatus and methods for performing electrochemical reactions
US20120261274A1 (en) 2009-05-29 2012-10-18 Life Technologies Corporation Methods and apparatus for measuring analytes
US9376713B2 (en) * 2009-09-23 2016-06-28 The Board Of Trustees Of The University Of Illinois Label free detection of nucleic acid amplification
US9487839B2 (en) * 2009-09-29 2016-11-08 Case Western Reserve University Method for detecting single nucleotide polymorphisms
WO2011071923A2 (en) * 2009-12-07 2011-06-16 Illumina, Inc. Multi-sample indexing for multiplex genotyping
US9315857B2 (en) 2009-12-15 2016-04-19 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse label-tags
US8835358B2 (en) 2009-12-15 2014-09-16 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
PT2531880T (en) 2010-02-01 2016-10-31 Illumina Inc Focusing methods and optical systems and assemblies using the same
US8481903B2 (en) 2010-03-06 2013-07-09 Alexander Triener Systems, methods, and apparatuses including a moveable optical component for detecting optical signals from a sample
US8951940B2 (en) 2010-04-01 2015-02-10 Illumina, Inc. Solid-phase clonal amplification and related methods
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
US20190300945A1 (en) 2010-04-05 2019-10-03 Prognosys Biosciences, Inc. Spatially Encoded Biological Assays
WO2011127099A1 (en) 2010-04-05 2011-10-13 Prognosys Biosciences, Inc. Spatially encoded biological assays
US9353412B2 (en) 2010-06-18 2016-05-31 Illumina, Inc. Conformational probes and methods for sequencing nucleic acids
JP5952813B2 (en) 2010-06-30 2016-07-13 ライフ テクノロジーズ コーポレーション Method and apparatus for testing ISFET arrays
JP2013533482A (en) 2010-06-30 2013-08-22 ライフ テクノロジーズ コーポレーション Ion-sensitive charge storage circuit and method
CN103392233B (en) 2010-06-30 2016-08-24 生命科技公司 Array column integrator
US11307166B2 (en) 2010-07-01 2022-04-19 Life Technologies Corporation Column ADC
TWI527245B (en) 2010-07-03 2016-03-21 生命技術公司 Chemically sensitive sensor with lightly doped drains
US20120028826A1 (en) * 2010-07-27 2012-02-02 Affymetrix, Inc. Methods and Compositions for Analysis of Nucleic Acids
EP2617061B1 (en) 2010-09-15 2021-06-30 Life Technologies Corporation Methods and apparatus for measuring analytes
CN103154273A (en) 2010-09-21 2013-06-12 群体遗传学科技有限公司 Increasing confidence of allele calls with molecular counting
US8796036B2 (en) 2010-09-24 2014-08-05 Life Technologies Corporation Method and system for delta double sampling
WO2012050920A1 (en) 2010-09-29 2012-04-19 Illumina, Inc. Compositions and methods for sequencing nucleic acids
WO2012055929A1 (en) 2010-10-26 2012-05-03 Illumina, Inc. Sequencing methods
US9074251B2 (en) 2011-02-10 2015-07-07 Illumina, Inc. Linking sequence reads using paired code tags
EP2649196B1 (en) * 2010-12-08 2015-03-18 Imec A biosensor using impedimetric real-time monitoring
CN103261416B (en) 2010-12-16 2016-08-10 安捷伦科技有限公司 Utilize the method for attachment of eucaryon tRNA ligase
US8951781B2 (en) 2011-01-10 2015-02-10 Illumina, Inc. Systems, methods, and apparatuses to image a sample for biological or chemical analysis
GB201101200D0 (en) 2011-01-24 2011-03-09 King S College Method
EP2670894B1 (en) 2011-02-02 2017-11-29 University Of Washington Through Its Center For Commercialization Massively parallel continguity mapping
GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product
US8478545B2 (en) 2011-06-03 2013-07-02 Agilent Technologies, Inc. Identification of aberrant microarray features
EP2718465B1 (en) 2011-06-09 2022-04-13 Illumina, Inc. Method of making an analyte array
US9435812B2 (en) 2011-08-31 2016-09-06 Ventana Medical Systems, Inc. Expression of ETS related gene (ERG) and phosphatase and tensin homolog (PTEN) correlates with prostate cancer capsular penetration
DE102011054101A1 (en) * 2011-09-30 2013-04-04 Albert-Ludwigs-Universität Freiburg Method for the spatial arrangement of sample fragments for amplification and immobilization for further derivatizations
US9566560B2 (en) 2011-10-06 2017-02-14 Illumina, Inc. Array domains having rotated patterns
EP3305400A3 (en) 2011-10-28 2018-06-06 Illumina, Inc. Microarray fabrication system and method
US9970984B2 (en) 2011-12-01 2018-05-15 Life Technologies Corporation Method and apparatus for identifying defects in a chemical sensor array
US8821798B2 (en) 2012-01-19 2014-09-02 Life Technologies Corporation Titanium nitride as sensing layer for microwell structure
US8747748B2 (en) 2012-01-19 2014-06-10 Life Technologies Corporation Chemical sensor with conductive cup-shaped sensor surface
CN104364392B (en) 2012-02-27 2018-05-25 赛卢拉研究公司 For the composition and kit of numerator counts
WO2013130512A2 (en) 2012-02-27 2013-09-06 The University Of North Carolina At Chapel Hill Methods and uses for molecular tags
EP2820155B1 (en) 2012-02-28 2017-07-26 Population Genetics Technologies Ltd. Method for attaching a counter sequence to a nucleic acid sample
CA3138752C (en) 2012-04-03 2024-02-06 Illumina, Inc. Integrated optoelectronic read head and fluidic cartridge useful for nucleic acid sequencing
US8786331B2 (en) 2012-05-29 2014-07-22 Life Technologies Corporation System for reducing noise in a chemical sensor array
US9012022B2 (en) 2012-06-08 2015-04-21 Illumina, Inc. Polymer coatings
WO2013187628A1 (en) * 2012-06-11 2013-12-19 Seegene, Inc. Detection of target nucleic acid sequence by pto cleavage and extension-dependent transcription
US8895249B2 (en) 2012-06-15 2014-11-25 Illumina, Inc. Kinetic exclusion amplification of nucleic acid libraries
US20160040229A1 (en) 2013-08-16 2016-02-11 Guardant Health, Inc. Systems and methods to detect rare mutations and copy number variation
US10876152B2 (en) 2012-09-04 2020-12-29 Guardant Health, Inc. Systems and methods to detect rare mutations and copy number variation
IL305303A (en) 2012-09-04 2023-10-01 Guardant Health Inc Systems and methods to detect rare mutations and copy number variation
US11913065B2 (en) 2012-09-04 2024-02-27 Guardent Health, Inc. Systems and methods to detect rare mutations and copy number variation
US9181583B2 (en) 2012-10-23 2015-11-10 Illumina, Inc. HLA typing using selective amplification and sequencing
US9080968B2 (en) 2013-01-04 2015-07-14 Life Technologies Corporation Methods and systems for point of use removal of sacrificial material
US9841398B2 (en) 2013-01-08 2017-12-12 Life Technologies Corporation Methods for manufacturing well structures for low-noise chemical sensors
US9683230B2 (en) 2013-01-09 2017-06-20 Illumina Cambridge Limited Sample preparation on a solid support
US8962366B2 (en) 2013-01-28 2015-02-24 Life Technologies Corporation Self-aligned well structures for low-noise chemical sensors
US9512422B2 (en) 2013-02-26 2016-12-06 Illumina, Inc. Gel patterned surfaces
CA2898456C (en) 2013-03-13 2020-11-10 Illumina, Inc. Methods and compositions for nucleic acid sequencing
US8963216B2 (en) 2013-03-13 2015-02-24 Life Technologies Corporation Chemical sensor with sidewall spacer sensor surface
US8841217B1 (en) 2013-03-13 2014-09-23 Life Technologies Corporation Chemical sensor with protruded sensor surface
US20160023208A1 (en) 2013-03-13 2016-01-28 Illumina, Inc. Multilayer fluidic devices and methods for their fabrication
US20140274747A1 (en) 2013-03-15 2014-09-18 Illumina, Inc. Super resolution imaging
US9835585B2 (en) 2013-03-15 2017-12-05 Life Technologies Corporation Chemical sensor with protruded sensor surface
WO2014149779A1 (en) 2013-03-15 2014-09-25 Life Technologies Corporation Chemical device with thin conductive element
US9193998B2 (en) 2013-03-15 2015-11-24 Illumina, Inc. Super resolution imaging
CN105264366B (en) 2013-03-15 2019-04-16 生命科技公司 Chemical sensor with consistent sensor surface area
EP3388442A1 (en) 2013-03-15 2018-10-17 Illumina Cambridge Limited Modified nucleosides or nucleotides
CN105283758B (en) 2013-03-15 2018-06-05 生命科技公司 Chemical sensor with consistent sensor surface area
US20140274749A1 (en) 2013-03-15 2014-09-18 Affymetrix, Inc. Systems and Methods for SNP Characterization and Identifying off Target Variants
US9116117B2 (en) 2013-03-15 2015-08-25 Life Technologies Corporation Chemical sensor with sidewall sensor surface
ES2751402T3 (en) 2013-03-27 2020-03-31 Bluegnome Ltd Aneuploidy risk assessment
US20140336063A1 (en) 2013-05-09 2014-11-13 Life Technologies Corporation Windowed Sequencing
US10458942B2 (en) 2013-06-10 2019-10-29 Life Technologies Corporation Chemical sensor array having multiple sensors per well
CN111500680A (en) 2013-06-25 2020-08-07 普罗格诺西斯生物科学公司 Method and system for detecting spatial distribution of biological targets in a sample
DK3017065T3 (en) 2013-07-01 2018-11-26 Illumina Inc Catalyst-free Surface functionalization and polymer grafting
WO2015002789A1 (en) 2013-07-03 2015-01-08 Illumina, Inc. Sequencing by orthogonal synthesis
WO2015031691A1 (en) 2013-08-28 2015-03-05 Cellular Research, Inc. Massively parallel single cell analysis
AU2014312043A1 (en) 2013-08-30 2016-02-25 Illumina France Manipulation of droplets on hydrophilic or variegated-hydrophilic surfaces
US9352315B2 (en) 2013-09-27 2016-05-31 Taiwan Semiconductor Manufacturing Company, Ltd. Method to produce chemical pattern in micro-fluidic structure
US9582877B2 (en) 2013-10-07 2017-02-28 Cellular Research, Inc. Methods and systems for digitally counting features on arrays
US10540783B2 (en) * 2013-11-01 2020-01-21 Illumina, Inc. Image analysis useful for patterned objects
AU2014364898B2 (en) 2013-12-19 2018-07-26 Illumina, Inc. Substrates comprising nano-patterning surfaces and methods of preparing thereof
AU2014364926B2 (en) 2013-12-20 2017-12-14 Illumina, Inc. Preserving genomic connectivity information in fragmented genomic DNA samples
CN106062212A (en) 2013-12-23 2016-10-26 伊鲁米那股份有限公司 Structured substrates for improving detection of light emissions and methods relating to the same
AU2014369841B2 (en) 2013-12-28 2019-01-24 Guardant Health, Inc. Methods and systems for detecting genetic variants
CN105917006B (en) 2014-01-16 2021-03-09 伊鲁米那股份有限公司 Amplicon preparation and sequencing on solid support
AU2015206336B2 (en) 2014-01-16 2020-01-23 Illumina, Inc. Gene expression panel for prognosis of prostate cancer recurrence
US9677132B2 (en) 2014-01-16 2017-06-13 Illumina, Inc. Polynucleotide modification on solid support
KR20150092585A (en) * 2014-02-05 2015-08-13 한국전자통신연구원 DNA data compression Method and Apparatus based on binary image
JP6510029B2 (en) 2014-07-15 2019-05-08 イラミーナ インコーポレーテッド Biochemically operated electronic devices
GB201414098D0 (en) 2014-08-08 2014-09-24 Illumina Cambridge Ltd Modified nucleotide linkers
US9982250B2 (en) 2014-08-21 2018-05-29 Illumina Cambridge Limited Reversible surface functionalization
SG10201903408VA (en) 2014-10-17 2019-05-30 Illumina Cambridge Ltd Contiguity preserving transposition
AU2015340767B2 (en) 2014-10-31 2020-01-30 Illumina Cambridge Limited Novel polymers and DNA copolymer coatings
EP3234187B1 (en) 2014-12-15 2021-02-17 Illumina, Inc. Method for single molecular placement on a substrate
US10077472B2 (en) 2014-12-18 2018-09-18 Life Technologies Corporation High data rate integrated circuit with power management
US10605767B2 (en) 2014-12-18 2020-03-31 Life Technologies Corporation High data rate integrated circuit with transmitter configuration
US10379079B2 (en) 2014-12-18 2019-08-13 Life Technologies Corporation Methods and apparatus for measuring analytes using large scale FET arrays
EP3766988B1 (en) 2015-02-19 2024-02-14 Becton, Dickinson and Company High-throughput single-cell analysis combining proteomic and genomic information
EP3262192B1 (en) 2015-02-27 2020-09-16 Becton, Dickinson and Company Spatially addressable molecular barcoding
US9976174B2 (en) 2015-03-24 2018-05-22 Illumina Cambridge Limited Methods, carrier assemblies, and systems for imaging samples for biological or chemical analysis
JP2018509915A (en) 2015-03-30 2018-04-12 セルラー リサーチ, インコーポレイテッド Methods and compositions for combinatorial bar coding
ES2955488T3 (en) 2015-04-10 2023-12-01 Spatial Transcriptomics Ab Multiplex analysis of biological specimens of spatially distinguished nucleic acids
US10900030B2 (en) 2015-04-14 2021-01-26 Illumina, Inc. Structured substrates for improving detection of light emissions and methods relating to the same
US11390914B2 (en) 2015-04-23 2022-07-19 Becton, Dickinson And Company Methods and compositions for whole transcriptome amplification
RU2724998C2 (en) 2015-05-11 2020-06-29 Иллюмина, Инк. Platform for detecting and analyzing therapeutic agents
CA2987397C (en) 2015-05-29 2022-03-01 Illumina, Inc. Sample carrier and assay system for conducting designated reactions
WO2016196229A1 (en) 2015-06-01 2016-12-08 Cellular Research, Inc. Methods for rna quantification
EP3320399A1 (en) 2015-07-07 2018-05-16 Illumina, Inc. Selective surface patterning via nanoimprinting
ES2945607T3 (en) 2015-07-17 2023-07-04 Illumina Inc Polymer sheets for sequencing applications
AU2016298541B2 (en) 2015-07-30 2019-10-31 Illumina, Inc. Orthogonal deblocking of nucleotides
CN108474805A (en) 2015-08-24 2018-08-31 亿明达股份有限公司 For accumulator and flow control system in biological and chemical setting-out line road
WO2017037078A1 (en) 2015-09-02 2017-03-09 Illumina Cambridge Limited Systems and methods of improving droplet operations in fluidic systems
EP3347465B1 (en) 2015-09-11 2019-06-26 Cellular Research, Inc. Methods and compositions for nucleic acid library normalization
US11319593B2 (en) 2015-12-17 2022-05-03 Illumina, Inc. Distinguishing methylation levels in complex biological samples
SG11201805119QA (en) 2015-12-17 2018-07-30 Guardant Health Inc Methods to determine tumor gene copy number by analysis of cell-free dna
CA3008031A1 (en) 2016-01-11 2017-07-20 Illumina Singapore Pte Ltd Detection apparatus having a microfluorometer, a fluidic system, and a flow cell latch clamp module
ES2861350T3 (en) 2016-03-28 2021-10-06 Illumina Inc Multi-plane microarrays
US10822643B2 (en) 2016-05-02 2020-11-03 Cellular Research, Inc. Accurate molecular barcoding
CA3021915A1 (en) 2016-05-18 2017-11-23 Illumina, Inc. Self-assembled patterning using patterned hydrophobic surfaces
US10301677B2 (en) 2016-05-25 2019-05-28 Cellular Research, Inc. Normalization of nucleic acid libraries
JP7046007B2 (en) 2016-05-26 2022-04-01 ベクトン・ディキンソン・アンド・カンパニー How to adjust the molecular label count
US10202641B2 (en) 2016-05-31 2019-02-12 Cellular Research, Inc. Error correction in amplification of samples
US10640763B2 (en) 2016-05-31 2020-05-05 Cellular Research, Inc. Molecular indexing of internal sequences
CN115060911A (en) 2016-09-26 2022-09-16 贝克顿迪金森公司 Measurement of protein expression using reagents with barcoded oligonucleotide sequences
WO2018064116A1 (en) 2016-09-28 2018-04-05 Illumina, Inc. Methods and systems for data compression
FI3522713T3 (en) 2016-10-03 2023-01-13 Fluorescent detection of amines and hydrazines and assaying methods thereof
WO2018089378A1 (en) 2016-11-08 2018-05-17 Cellular Research, Inc. Methods for expression profile classification
US11608497B2 (en) 2016-11-08 2023-03-21 Becton, Dickinson And Company Methods for cell label classification
GB201619458D0 (en) 2016-11-17 2017-01-04 Spatial Transcriptomics Ab Method for spatial tagging and analysing nucleic acids in a biological specimen
GB201704754D0 (en) 2017-01-05 2017-05-10 Illumina Inc Kinetic exclusion amplification of nucleic acid libraries
CN110573253B (en) 2017-01-13 2021-11-02 赛卢拉研究公司 Hydrophilic coating for fluid channels
SG11201906567YA (en) 2017-01-20 2019-08-27 Omniome Inc Allele-specific capture of nucleic acids
US9932631B1 (en) 2017-09-11 2018-04-03 Omniome, Inc. Genotyping by polymerase binding
SG11201906569XA (en) 2017-01-20 2019-08-27 Omniome Inc Genotyping by polymerase binding
WO2018144531A1 (en) 2017-02-01 2018-08-09 Illumina, Inc. System and method with fiducials responding to multiple excitation frequencies
GB201701689D0 (en) 2017-02-01 2017-03-15 Illumia Inc System and method with fiducials of non-closed shapes
GB201701691D0 (en) 2017-02-01 2017-03-15 Illumina Inc System and method with reflective fiducials
CN110382708A (en) 2017-02-01 2019-10-25 赛卢拉研究公司 Selective amplification is carried out using blocking property oligonucleotides
GB201701688D0 (en) 2017-02-01 2017-03-15 Illumia Inc System and method with fiducials in non-recliner layouts
GB201701686D0 (en) 2017-02-01 2017-03-15 Illunina Inc System & method with fiducials having offset layouts
WO2018152162A1 (en) 2017-02-15 2018-08-23 Omniome, Inc. Distinguishing sequences by detecting polymerase dissociation
US10161003B2 (en) 2017-04-25 2018-12-25 Omniome, Inc. Methods and apparatus that increase sequencing-by-binding efficiency
AU2018281745B2 (en) 2017-06-05 2022-05-19 Becton, Dickinson And Company Sample indexing for single cells
EP4289967A2 (en) 2017-08-01 2023-12-13 Illumina, Inc. Spatial indexing of genetic material and library preparation using hydrogel beads and flow cells
JP7032452B2 (en) 2017-08-01 2022-03-08 イルミナ インコーポレイテッド Hydrogel beads for nucleotide sequencing
CN111182972B (en) 2017-08-15 2022-03-08 欧姆尼奥姆股份有限公司 Scanning device and method for detecting chemical and biological analytes
US11193166B2 (en) 2017-10-19 2021-12-07 Omniome, Inc. Simultaneous background reduction and complex stabilization in binding assay workflows
KR20220123327A (en) 2018-02-13 2022-09-06 일루미나, 인코포레이티드 DNA sequencing using hydrogel beads
EP3782158A1 (en) 2018-04-19 2021-02-24 Omniome, Inc. Improving accuracy of base calls in nucleic acid sequencing methods
KR20230005427A (en) 2018-04-20 2023-01-09 일루미나, 인코포레이티드 Methods of encapsulating single cells, the encapsulated cells and uses thereof
CN112567047A (en) 2018-04-26 2021-03-26 欧姆尼欧美公司 Methods and compositions for stabilizing nucleic acid-nucleotide-polymerase complexes
CN112243461A (en) 2018-05-03 2021-01-19 贝克顿迪金森公司 Molecular barcoding at opposite transcript ends
ES2945191T3 (en) 2018-05-03 2023-06-29 Becton Dickinson Co High-throughput multi-omics sample analysis
WO2019231568A1 (en) 2018-05-31 2019-12-05 Omniome, Inc. Increased signal to noise in nucleic acid sequencing
CA3107165A1 (en) 2018-07-24 2020-01-30 Omniome, Inc. Serial formation of ternary complex species
US11519033B2 (en) 2018-08-28 2022-12-06 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample
EP3844308A1 (en) 2018-08-28 2021-07-07 10X Genomics, Inc. Resolving spatial arrays
US20230323447A1 (en) 2018-08-28 2023-10-12 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample
US10731141B2 (en) 2018-09-17 2020-08-04 Omniome, Inc. Engineered polymerases for improved sequencing
EP3861134A1 (en) 2018-10-01 2021-08-11 Becton, Dickinson and Company Determining 5' transcript sequences
KR20210084440A (en) 2018-10-26 2021-07-07 일루미나, 인코포레이티드 Control of Polymer Beads for DNA Processing
WO2020101795A1 (en) 2018-11-15 2020-05-22 Omniome, Inc. Electronic detection of nucleic acid structure
EP4293126A3 (en) 2018-11-30 2024-01-17 Illumina, Inc. Analysis of multiple analytes using a single assay
US10710076B2 (en) 2018-12-04 2020-07-14 Omniome, Inc. Mixed-phase fluids for nucleic acid sequencing and other analytical assays
JP2022511207A (en) 2018-12-05 2022-01-31 イルミナ ケンブリッジ リミテッド Methods and compositions for cluster formation by bridge amplification
US20220025446A1 (en) 2018-12-10 2022-01-27 10X Genomics, Inc. Methods of using master / copy arrays for spatial detection
GB201820300D0 (en) 2018-12-13 2019-01-30 10X Genomics Inc Method for spatial tagging and analysing genomic DNA in a biological specimen
EP3894552A1 (en) 2018-12-13 2021-10-20 Becton, Dickinson and Company Selective extension in single cell whole transcriptome analysis
GB201820341D0 (en) 2018-12-13 2019-01-30 10X Genomics Inc Method for transposase-mediated spatial tagging and analysing genomic DNA in a biological specimen
CA3103633A1 (en) 2018-12-18 2020-06-25 Illumina Cambridge Limited Methods and compositions for paired end sequencing using a single surface primer
MX2020013379A (en) 2018-12-19 2021-04-28 Illumina Inc Methods for improving polynucleotide cluster clonality priority.
WO2020132350A2 (en) 2018-12-20 2020-06-25 Omniome, Inc. Temperature control for analysis of nucleic acids and other analytes
US11293061B2 (en) 2018-12-26 2022-04-05 Illumina Cambridge Limited Sequencing methods using nucleotides with 3′ AOM blocking group
US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis
US11371076B2 (en) 2019-01-16 2022-06-28 Becton, Dickinson And Company Polymerase chain reaction normalization through primer titration
EP4242322A3 (en) 2019-01-23 2023-09-20 Becton, Dickinson and Company Oligonucleotides associated with antibodies
US11499189B2 (en) 2019-02-14 2022-11-15 Pacific Biosciences Of California, Inc. Mitigating adverse impacts of detection systems on nucleic acids and other biological analytes
EP3927467A4 (en) 2019-02-20 2022-12-14 Pacific Biosciences of California, Inc. Scanning apparatus and methods for detecting chemical and biological analytes
EP3931354A1 (en) 2019-02-28 2022-01-05 10X Genomics, Inc. Profiling of biological analytes with spatially barcoded oligonucleotide arrays
EP3938538A1 (en) 2019-03-15 2022-01-19 10X Genomics, Inc. Methods for using spatial arrays for single cell sequencing
NL2023314B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based quality scoring
NL2023312B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based base calling
NL2023316B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based sequencing
NL2023311B9 (en) 2019-03-21 2021-03-12 Illumina Inc Artificial intelligence-based generation of sequencing metadata
WO2020191387A1 (en) 2019-03-21 2020-09-24 Illumina, Inc. Artificial intelligence-based base calling
US11210554B2 (en) 2019-03-21 2021-12-28 Illumina, Inc. Artificial intelligence-based generation of sequencing metadata
NL2023310B1 (en) 2019-03-21 2020-09-28 Illumina Inc Training data generation for artificial intelligence-based sequencing
WO2020198071A1 (en) 2019-03-22 2020-10-01 10X Genomics, Inc. Three-dimensional spatial analysis
US11421271B2 (en) 2019-03-28 2022-08-23 Illumina Cambridge Limited Methods and compositions for nucleic acid sequencing using photoswitchable labels
US11593649B2 (en) 2019-05-16 2023-02-28 Illumina, Inc. Base calling using convolutions
US11644406B2 (en) 2019-06-11 2023-05-09 Pacific Biosciences Of California, Inc. Calibrated focus sensing
BR112021012755A2 (en) 2019-07-12 2022-04-26 Illumina Cambridge Ltd Compositions and methods for preparing nucleic acid sequencing libraries using crispr/cas9 immobilized on a solid support
WO2021011803A1 (en) 2019-07-16 2021-01-21 Omniome, Inc. Synthetic nucleic acids having non-natural structures
US10656368B1 (en) 2019-07-24 2020-05-19 Omniome, Inc. Method and system for biological imaging using a wide field objective lens
US11180520B2 (en) 2019-09-10 2021-11-23 Omniome, Inc. Reversible modifications of nucleotides
EP4038546A1 (en) 2019-10-01 2022-08-10 10X Genomics, Inc. Systems and methods for identifying morphological patterns in tissue samples
WO2021076152A1 (en) 2019-10-18 2021-04-22 Omniome, Inc. Methods and compositions for capping nucleic acids
WO2021092433A2 (en) 2019-11-08 2021-05-14 10X Genomics, Inc. Enhancing specificity of analyte binding
US20210139867A1 (en) 2019-11-08 2021-05-13 Omniome, Inc. Engineered polymerases for improved sequencing by binding
EP4055185A1 (en) 2019-11-08 2022-09-14 10X Genomics, Inc. Spatially-tagged analyte capture agents for analyte multiplexing
JP2023500679A (en) 2019-11-08 2023-01-10 ベクトン・ディキンソン・アンド・カンパニー Using random priming to obtain full-length V(D)J information for immune repertoire sequencing
CN115004260A (en) 2019-11-18 2022-09-02 10X基因组学有限公司 System and method for tissue classification
US20210155982A1 (en) 2019-11-21 2021-05-27 10X Genomics, Inc. Pipeline for spatial analysis of analytes
CA3158891A1 (en) 2019-11-22 2021-05-27 Neil Ira WEISENFELD Systems and methods for spatial analysis of analytes using fiducial alignment
SG11202106899SA (en) 2019-12-23 2021-09-29 10X Genomics Inc Methods for spatial analysis using rna-templated ligation
EP4090763A1 (en) 2020-01-13 2022-11-23 Becton Dickinson and Company Methods and compositions for quantitation of proteins and rna
US11732299B2 (en) 2020-01-21 2023-08-22 10X Genomics, Inc. Spatial assays with perturbed cells
US11702693B2 (en) 2020-01-21 2023-07-18 10X Genomics, Inc. Methods for printing cells and generating arrays of barcoded cells
US11821035B1 (en) 2020-01-29 2023-11-21 10X Genomics, Inc. Compositions and methods of making gene expression libraries
US11898205B2 (en) 2020-02-03 2024-02-13 10X Genomics, Inc. Increasing capture efficiency of spatial assays
WO2021158511A1 (en) 2020-02-04 2021-08-12 Omniome, Inc. Flow cells and methods for their manufacture and use
US11732300B2 (en) 2020-02-05 2023-08-22 10X Genomics, Inc. Increasing efficiency of spatial analysis in a biological sample
US11835462B2 (en) 2020-02-11 2023-12-05 10X Genomics, Inc. Methods and compositions for partitioning a biological sample
US20210265018A1 (en) 2020-02-20 2021-08-26 Illumina, Inc. Knowledge Distillation and Gradient Pruning-Based Compression of Artificial Intelligence-Based Base Caller
JP2023514749A (en) 2020-02-21 2023-04-07 10エックス ジェノミクス インコーポレイテッド Methods and compositions for integrated in situ spatial assays
US11891654B2 (en) 2020-02-24 2024-02-06 10X Genomics, Inc. Methods of making gene expression libraries
CA3173798A1 (en) 2020-03-03 2021-09-10 Pacific Biosciences Of California, Inc. Methods and compositions for sequencing double stranded nucleic acids
US11768175B1 (en) 2020-03-04 2023-09-26 10X Genomics, Inc. Electrophoretic methods for spatial analysis
CN115916999A (en) 2020-04-22 2023-04-04 10X基因组学有限公司 Methods for spatial analysis using targeted RNA depletion
CN115836135A (en) 2020-05-05 2023-03-21 加利福尼亚太平洋生物科学股份有限公司 Compositions and methods for modifying polymerase-nucleic acid complexes
US11188778B1 (en) 2020-05-05 2021-11-30 Illumina, Inc. Equalization-based image processing and spatial crosstalk attenuator
EP4150118A1 (en) 2020-05-14 2023-03-22 Becton Dickinson and Company Primers for immune repertoire profiling
EP4153775A1 (en) 2020-05-22 2023-03-29 10X Genomics, Inc. Simultaneous spatio-temporal measurement of gene expression and cellular activity
EP4153776A1 (en) 2020-05-22 2023-03-29 10X Genomics, Inc. Spatial analysis to detect sequence variants
WO2021242834A1 (en) 2020-05-26 2021-12-02 10X Genomics, Inc. Method for resetting an array
WO2021247568A1 (en) 2020-06-02 2021-12-09 10X Genomics, Inc. Spatial trancriptomics for antigen-receptors
WO2021247543A2 (en) 2020-06-02 2021-12-09 10X Genomics, Inc. Nucleic acid library methods
EP4162074A1 (en) 2020-06-08 2023-04-12 10X Genomics, Inc. Methods of determining a surgical margin and methods of use thereof
WO2021252591A1 (en) 2020-06-10 2021-12-16 10X Genomics, Inc. Methods for determining a location of an analyte in a biological sample
CN115867560A (en) 2020-06-22 2023-03-28 伊鲁米纳剑桥有限公司 Nucleosides and nucleotides having 3' acetal capping groups
EP4172362A1 (en) 2020-06-25 2023-05-03 10X Genomics, Inc. Spatial analysis of dna methylation
AU2021299216A1 (en) 2020-06-30 2022-12-08 Illumina, Inc. Catalytically controlled sequencing by synthesis to produce scarless DNA
US11761038B1 (en) 2020-07-06 2023-09-19 10X Genomics, Inc. Methods for identifying a location of an RNA in a biological sample
US20220033900A1 (en) 2020-07-28 2022-02-03 Illumina Cambridge Limited Substituted coumarin dyes and uses as fluorescent labels
EP4192951A1 (en) 2020-08-06 2023-06-14 Illumina, Inc. Preparation of rna and dna sequencing libraries using bead-linked transposomes
CN116323971A (en) 2020-08-18 2023-06-23 Illumina公司 Sequence-specific targeted transposition and selection and sorting of nucleic acids
KR20230069135A (en) 2020-09-11 2023-05-18 일루미나 케임브리지 리미티드 Methods for Enriching Target Sequences in Sequencing Libraries Using Hairpin Adapters
US11827935B1 (en) 2020-11-19 2023-11-28 10X Genomics, Inc. Methods for spatial analysis using rolling circle amplification and detection probes
US11739443B2 (en) 2020-11-20 2023-08-29 Becton, Dickinson And Company Profiling of highly expressed and lowly expressed proteins
AU2021409136A1 (en) 2020-12-21 2023-06-29 10X Genomics, Inc. Methods, compositions, and systems for capturing probes and/or barcodes
CA3208854A1 (en) 2021-02-04 2022-08-11 Illumina, Inc. Long indexed-linked read generation on transposome bound beads
AU2022238446A1 (en) 2021-03-18 2023-09-07 10X Genomics, Inc. Multiplex capture of gene and protein expression from a biological sample
WO2022204032A1 (en) 2021-03-22 2022-09-29 Illumina Cambridge Limited Methods for improving nucleic acid cluster clonality
AU2022252197A1 (en) 2021-03-29 2023-09-21 Illumina, Inc. Improved methods of library preparation
KR20230163434A (en) 2021-03-29 2023-11-30 일루미나, 인코포레이티드 Compositions and methods for assessing DNA damage and normalizing amplicon size bias in libraries
IL307164A (en) 2021-03-31 2023-11-01 Illumina Inc Methods of preparing directional tagmentation sequencing libraries using transposon-based technology with unique molecular identifiers for error correction
KR20240009435A (en) 2021-05-20 2024-01-22 일루미나, 인코포레이티드 Compositions and methods for sequencing by synthesis
US20230016633A1 (en) 2021-06-15 2023-01-19 Illumina, Inc. Hydrogel-free surface functionalization for sequencing
WO2022272260A1 (en) 2021-06-23 2022-12-29 Illumina, Inc. Compositions, methods, kits, cartridges, and systems for sequencing reagents
WO2023003757A1 (en) 2021-07-19 2023-01-26 Illumina Software, Inc. Intensity extraction with interpolation and adaptation for base calling
US11455487B1 (en) 2021-10-26 2022-09-27 Illumina Software, Inc. Intensity extraction and crosstalk attenuation using interpolation and adaptation for base calling
WO2023004357A1 (en) 2021-07-23 2023-01-26 Illumina, Inc. Methods for preparing substrate surface for dna sequencing
EP4196605A1 (en) 2021-09-01 2023-06-21 10X Genomics, Inc. Methods, compositions, and kits for blocking a capture probe on a spatial array
WO2023038859A1 (en) 2021-09-09 2023-03-16 Nautilus Biotechnology, Inc. Characterization and localization of protein modifications
WO2023049215A1 (en) 2021-09-22 2023-03-30 Illumina, Inc. Compressed state-based base calling
WO2023069927A1 (en) 2021-10-20 2023-04-27 Illumina, Inc. Methods for capturing library dna for sequencing
US20230215515A1 (en) 2021-12-23 2023-07-06 Illumina Software, Inc. Facilitating secure execution of external workflows for genomic sequencing diagnostics
WO2023122363A1 (en) 2021-12-23 2023-06-29 Illumina Software, Inc. Dynamic graphical status summaries for nucelotide sequencing
WO2023129764A1 (en) 2021-12-29 2023-07-06 Illumina Software, Inc. Automatically switching variant analysis model versions for genomic analysis applications
CA3223362A1 (en) 2022-01-20 2023-07-27 Xiaolin Wu Methods of detecting methylcytosine and hydroxymethylcytosine by sequencing
WO2023183937A1 (en) 2022-03-25 2023-09-28 Illumina, Inc. Sequence-to-sequence base calling
CA3223115A1 (en) 2022-03-28 2023-10-05 Xiaolin Wu Labeled avidin and methods for sequencing
US20230313292A1 (en) 2022-03-29 2023-10-05 Illumina Cambridge Limited Chromenoquinoline dyes and uses in sequencing
WO2023196572A1 (en) 2022-04-07 2023-10-12 Illumina Singapore Pte. Ltd. Altered cytidine deaminases and methods of use
WO2023209606A1 (en) 2022-04-29 2023-11-02 Illumina Cambridge Limited Methods and systems for encapsulating lyophilised microspheres
US20230383342A1 (en) 2022-05-31 2023-11-30 Illumina Cambridge Limited Compositions and methods for nucleic acid sequencing
US20230407386A1 (en) 2022-06-09 2023-12-21 Illumina, Inc. Dependence of base calling on flow cell tilt
US20230416279A1 (en) 2022-06-28 2023-12-28 Illumina Cambridge Limited Fluorescent dyes containing fused tetracyclic bis-boron heterocycle and uses in sequencing
WO2024039516A1 (en) 2022-08-19 2024-02-22 Illumina, Inc. Third dna base pair site-specific dna detection

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5503980A (en) * 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization

Family Cites Families (92)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4200110A (en) 1977-11-28 1980-04-29 United States Of America Fiber optic pH probe
US4499052A (en) 1982-08-30 1985-02-12 Becton, Dickinson And Company Apparatus for distinguishing multiple subpopulations of cells
US4682895A (en) 1985-08-06 1987-07-28 Texas A&M University Fiber optic probe for quantification of colorimetric reactions
US4798738A (en) 1986-10-10 1989-01-17 Cardiovascular Devices, Inc. Micro sensor
US4824789B1 (en) 1986-10-10 1996-08-13 Minnesota Mining & Mfg Gas sensor
US4822746A (en) 1986-06-25 1989-04-18 Trustees Of Tufts College Radiative and non-radiative energy transfer and absorbance modulated fluorescence detection methods and sensors
US5143853A (en) 1986-06-25 1992-09-01 Trustees Of Tufts College Absorbance modulated fluorescence detection methods and sensors
US5254477A (en) 1986-06-25 1993-10-19 Trustees Of Tufts College Flourescence intramolecular energy transfer conjugate compositions and detection methods
US5114864A (en) 1986-06-25 1992-05-19 Trustees Of Tufts College Fiber optic sensors, apparatus, and detection methods using fluid erodible controlled release polymers for delivery of reagent formulations
US5252494A (en) 1986-06-25 1993-10-12 Trustees Of Tufts College Fiber optic sensors, apparatus, and detection methods using controlled release polymers and reagent formulations held within a polymeric reaction matrix
ES2025556T3 (en) 1986-12-01 1992-04-01 Molecular Biosystems, Inc. METHOD OF THE HYBRIDIZATION TEST TO DETECT A SPECIFIC DIANE POLYNUCLEOTIDIC SEQUENCE.
SE458968B (en) 1987-06-16 1989-05-22 Wallac Oy BIOSPECIFIC ANALYTICAL PROCEDURE FOR MULTIPLE ANALYTICS WHICH DO NOT INCLUDE PARTICULAR COATING AND LABELING WITH FLUORESCING LABEL SUBSTANCES
US5194300A (en) 1987-07-15 1993-03-16 Cheung Sau W Methods of making fluorescent microspheres
US5132242A (en) 1987-07-15 1992-07-21 Cheung Sau W Fluorescent microspheres and methods of using them
US4785814A (en) 1987-08-11 1988-11-22 Cordis Corporation Optical probe for measuring pH and oxygen in blood and employing a composite membrane
US5002867A (en) 1988-04-25 1991-03-26 Macevicz Stephen C Nucleic acid sequence determination by multiple mixed oligonucleotide probes
NO164622C (en) 1988-05-11 1990-10-24 Tore Lindmo BINAER IMMUNOMETRIC PARTICLE-BASED METHOD FOR MEASURING SPECIFIC SERUM ANTIGENS USING LIQUID FLOW MICROPHOTOMETRY AND A PREPARED TARGET SET UP THEREOF.
US5575849A (en) 1988-11-25 1996-11-19 Canon Kabushiki Kaisha Apparatus for producing a substrate having a surface with a plurality of spherical dimples for photoconductive members
EP0392546A3 (en) 1989-04-14 1991-09-11 Ro Institut Za Molekularnu Genetiku I Geneticko Inzenjerstvo Process for determination of a complete or a partial contents of very short sequences in the samples of nucleic acids connected to the discrete particles of microscopic size by hybridization with oligonucleotide probes
US5302509A (en) 1989-08-14 1994-04-12 Beckman Instruments, Inc. Method for sequencing polynucleotides
US5326692B1 (en) 1992-05-13 1996-04-30 Molecular Probes Inc Fluorescent microparticles with controllable enhanced stokes shift
US5494810A (en) 1990-05-03 1996-02-27 Cornell Research Foundation, Inc. Thermostable ligase-mediated DNA amplifications system for the detection of genetic disease
EP0478319B1 (en) 1990-09-28 1997-04-02 Kabushiki Kaisha Toshiba Gene detection method
AU9115891A (en) 1990-11-14 1992-06-11 Siska Diagnostics, Inc. Non-isotopic detection of nucleic acids using a polystyrene support-based sandwich hybridization assay and compositions useful therefor
US5105305A (en) 1991-01-10 1992-04-14 At&T Bell Laboratories Near-field scanning optical microscope using a fluorescent probe
US5244636A (en) 1991-01-25 1993-09-14 Trustees Of Tufts College Imaging fiber optic array sensors, apparatus, and methods for concurrently detecting multiple analytes of interest in a fluid sample
US5250264A (en) 1991-01-25 1993-10-05 Trustees Of Tufts College Method of making imaging fiber optic sensors to concurrently detect multiple analytes of interest in a fluid sample
US5244813A (en) 1991-01-25 1993-09-14 Trustees Of Tufts College Fiber optic sensor, apparatus, and methods for detecting an organic analyte in a fluid or vapor sample
US5320814A (en) 1991-01-25 1994-06-14 Trustees Of Tufts College Fiber optic array sensors, apparatus, and methods for concurrently visualizing and chemically detecting multiple analytes of interest in a fluid sample
US5380489A (en) 1992-02-18 1995-01-10 Eastman Kodak Company Element and method for nucleic acid amplification and detection using adhered probes
DE69227112D1 (en) 1991-07-16 1998-10-29 Transmed Biotech Inc METHOD AND COMPOSITIONS FOR SIMULTANEOUS ANALYSIS OF A VARIETY OF ANALYZES
US5639603A (en) 1991-09-18 1997-06-17 Affymax Technologies N.V. Synthesizing and screening molecular diversity
DE69223980T2 (en) 1991-10-15 1998-05-28 Multilyte Ltd BINDING TEST USING A MARKED REAGENT
US6051380A (en) * 1993-11-01 2000-04-18 Nanogen, Inc. Methods and procedures for molecular biological analysis and diagnostics
US5888723A (en) 1992-02-18 1999-03-30 Johnson & Johnson Clinical Diagnostics, Inc. Method for nucleic acid amplification and detection using adhered probes
EP0565999A2 (en) 1992-04-16 1993-10-20 Siemens Aktiengesellschaft Optical coupling device for two groups of waveguides
AU681082B2 (en) * 1992-05-06 1997-08-21 Gen-Probe Incorporated Nucleic acid sequence amplification method, composition and kit
CA2115342C (en) 1992-06-17 2003-08-26 Robert B. Wallace A method of detecting and discriminating between nucleic acid sequences
DE69313611T2 (en) 1992-07-02 1998-01-08 Erkki Soini BIOS-SPECIFIC MULTIPARAMETER ANALYSIS PROCEDURE
DE69333502T2 (en) 1992-09-14 2005-04-14 Sri International, Menlo Park Up-converting reporter molecule for biological and other testing using laser excitation techniques
US5565324A (en) 1992-10-01 1996-10-15 The Trustees Of Columbia University In The City Of New York Complex combinatorial chemical libraries encoded with tags
US5541311A (en) * 1992-12-07 1996-07-30 Third Wave Technologies, Inc. Nucleic acid encoding synthesis-deficient thermostable DNA polymerase
US5298741A (en) 1993-01-13 1994-03-29 Trustees Of Tufts College Thin film fiber optic sensor array and apparatus for concurrent viewing and chemical sensing of a sample
CA2102884A1 (en) 1993-03-04 1994-09-05 James J. Wynne Dental procedures and apparatus using ultraviolet radiation
JP3302458B2 (en) 1993-08-31 2002-07-15 富士通株式会社 Integrated optical device and manufacturing method
WO1995014106A2 (en) * 1993-11-17 1995-05-26 Id Biomedical Corporation Cycling probe cleavage detection of nucleic acid sequences
US5494798A (en) 1993-12-09 1996-02-27 Gerdt; David W. Fiber optic evanscent wave sensor for immunoassay
US5496997A (en) 1994-01-03 1996-03-05 Pope; Edward J. A. Sensor incorporating an optical fiber and a solid porous inorganic microsphere
EP0754240B1 (en) 1994-02-07 2003-08-20 Beckman Coulter, Inc. Ligase/polymerase-mediated genetic bit analysis of single nucleotide polymorphisms and its use in genetic analysis
US5856083A (en) 1994-05-06 1999-01-05 Pharmacopeia, Inc. Lawn assay for compounds that affect enzyme activity or bind to target molecules
WO1996003212A1 (en) 1994-07-26 1996-02-08 Sydney Brenner Multidimensional conduit combinatorial library synthesis device
US5512490A (en) 1994-08-11 1996-04-30 Trustees Of Tufts College Optical sensor, optical sensing apparatus, and methods for detecting an analyte of interest using spectral recognition patterns
US5846719A (en) 1994-10-13 1998-12-08 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
WO1998053300A2 (en) 1997-05-23 1998-11-26 Lynx Therapeutics, Inc. System and apparaus for sequential processing of analytes
US5585069A (en) 1994-11-10 1996-12-17 David Sarnoff Research Center, Inc. Partitioned microelectronic and fluidic device array for clinical diagnostics and chemical synthesis
ATE215993T1 (en) * 1995-05-19 2002-04-15 Abbott Lab DETECTION OF NUCLEIC ACIDS WITH WIDE DYNAMIC RANGE USING AGGREGATE PRIMER SERIES
US5690894A (en) 1995-05-23 1997-11-25 The Regents Of The University Of California High density array fabrication and readout method for a fiber optic biosensor
US5656241A (en) 1995-09-07 1997-08-12 Optical Sensors Incorporated Method for manufacturing fiber optic sensors
WO1997014028A2 (en) 1995-10-11 1997-04-17 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method
GB9521159D0 (en) 1995-10-16 1995-12-20 Brint Norman T A weapon
US5854033A (en) * 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
US5633972A (en) 1995-11-29 1997-05-27 Trustees Of Tufts College Superresolution imaging fiber for subwavelength light energy generation and near-field optical microscopy
US5814524A (en) 1995-12-14 1998-09-29 Trustees Of Tufts College Optical sensor apparatus for far-field viewing and making optical analytical measurements at remote locations
EP1958955B1 (en) 1996-02-09 2013-09-04 Cornell Research Foundation, Inc. Spatially addressable oligonucleotide arrays and method of making the same
US5840256A (en) 1996-04-09 1998-11-24 David Sarnoff Research Center Inc. Plate for reaction system
DE69737883T2 (en) 1996-04-25 2008-03-06 Bioarray Solutions Ltd. LIGHT-REGULATED, ELECTROKINETIC COMPOSITION OF PARTICLES TO SURFACES
EP2369007B1 (en) 1996-05-29 2015-07-29 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
US5854684A (en) 1996-09-26 1998-12-29 Sarnoff Corporation Massively parallel detection
GB9620209D0 (en) 1996-09-27 1996-11-13 Cemu Bioteknik Ab Method of sequencing DNA
US5900481A (en) 1996-11-06 1999-05-04 Sequenom, Inc. Bead linkers for immobilizing nucleic acids to solid supports
CA2276462C (en) 1996-12-31 2007-06-12 Genometrix Incorporated Multiplexed molecular analysis system apparatus and method
US6327410B1 (en) 1997-03-14 2001-12-04 The Trustees Of Tufts College Target analyte sensors utilizing Microspheres
US6023540A (en) * 1997-03-14 2000-02-08 Trustees Of Tufts College Fiber optic sensor with encoded microspheres
DE69836012T2 (en) 1997-05-02 2007-04-05 Gen-Probe Inc., San Diego TWO-STEP HYBRIDIZATION AND INJECTION OF A POLYNUCLEOTIDE
US6406845B1 (en) 1997-05-05 2002-06-18 Trustees Of Tuft College Fiber optic biosensor for selectively detecting oligonucleotide species in a mixed fluid sample
AU756945B2 (en) 1997-05-23 2003-01-30 Bioarray Solutions Ltd Color-encoding and in-situ interrogation of matrix-coupled chemical compounds
US7115884B1 (en) 1997-10-06 2006-10-03 Trustees Of Tufts College Self-encoding fiber optic sensor
WO1999060170A1 (en) 1998-05-21 1999-11-25 Hyseq, Inc. Linear arrays of immobilized compounds and methods of using same
EP1090138A4 (en) 1998-06-24 2003-01-02 Glaxo Group Ltd Nucleotide detection method
ATE423314T1 (en) 1998-06-24 2009-03-15 Illumina Inc DECODING OF MATRIXED SENSORS BY MICROPARTICLES
US6908770B1 (en) 1998-07-16 2005-06-21 Board Of Regents, The University Of Texas System Fluid based analysis of multiple analytes by a sensor array
US5998175A (en) 1998-07-24 1999-12-07 Lumigen, Inc. Methods of synthesizing and amplifying polynucleotides by ligation of multiple oligomers
AU6131299A (en) 1998-08-26 2000-03-21 Trustees Of Tufts College Combinatorial polymer synthesis of sensors for polymer-based sensor arrays
US6429027B1 (en) 1998-12-28 2002-08-06 Illumina, Inc. Composite arrays utilizing microspheres
CA2359352C (en) 1999-02-09 2004-09-21 Illumina, Inc. Arrays comprising a fiducial and automated information processing in randomly ordered arrays
ATE462970T1 (en) 1999-02-09 2010-04-15 Illumina Inc SCREENING METHOD USING POROUS MICROBEADS AND COMPOSITIONS
CA2366459A1 (en) 1999-03-26 2000-10-05 Affymetrix, Inc. Universal arrays
EP1923472B1 (en) 1999-04-20 2012-04-11 Illumina, Inc. Detection of nucleic acid reactions on bead arrays
US6544732B1 (en) 1999-05-20 2003-04-08 Illumina, Inc. Encoding and decoding of array sensors utilizing nanocrystals
AU7569600A (en) 1999-05-20 2000-12-28 Illumina, Inc. Combinatorial decoding of random nucleic acid arrays
US20020051971A1 (en) 1999-05-21 2002-05-02 John R. Stuelpnagel Use of microfluidic systems in the detection of target analytes using microsphere arrays
DE60009323T2 (en) 1999-09-13 2005-02-10 Nugen Technologies, Inc., San Carlos METHODS AND COMPOSITIONS FOR LINEAR ISOTHERMAL AMPLIFICATION OF POLYNUCLEOTIDE SEQUENCES

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5503980A (en) * 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization

Cited By (132)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050089953A1 (en) * 1994-06-08 2005-04-28 Affymetrix, Inc. Bioarray chip reaction apparatus and its manufacture
US20040171054A1 (en) * 1994-06-08 2004-09-02 Affymetrix, Inc. Bioarray chip reaction apparatus and its manufacture
US20050158819A1 (en) * 1994-06-08 2005-07-21 Affymetrix, Inc. Bioarray chip reaction apparatus and its manufacture
US20050181403A1 (en) * 1995-06-07 2005-08-18 Affymetrix, Inc. Methods for making a device for concurrently processing multiple biological chip assays
US8460865B2 (en) 1998-06-24 2013-06-11 Illumina, Inc. Multiplex decoding of array sensors with microspheres
US9399795B2 (en) 1998-06-24 2016-07-26 Illumina, Inc. Multiplex decoding of array sensors with microspheres
US8628952B2 (en) 1998-12-28 2014-01-14 Illumina, Inc. Array kits and processing systems
US7901897B2 (en) 1998-12-28 2011-03-08 Illumina, Inc. Methods of making arrays
US8906626B2 (en) 2000-02-07 2014-12-09 Illumina, Inc. Multiplex nucleic acid reactions
US8288103B2 (en) 2000-02-07 2012-10-16 Illumina, Inc. Multiplex nucleic acid reactions
US8076063B2 (en) 2000-02-07 2011-12-13 Illumina, Inc. Multiplexed methylation detection methods
US8003354B2 (en) 2000-02-07 2011-08-23 Illumina, Inc. Multiplex nucleic acid reactions
US10837059B2 (en) 2000-02-07 2020-11-17 Illumina, Inc. Multiplex nucleic acid reactions
US9850536B2 (en) 2000-02-07 2017-12-26 Illumina, Inc. Multiplex nucleic acid reactions
US7846733B2 (en) 2000-06-26 2010-12-07 Nugen Technologies, Inc. Methods and compositions for transcription-based nucleic acid amplification
US7955794B2 (en) 2000-09-21 2011-06-07 Illumina, Inc. Multiplex nucleic acid reactions
US8334116B2 (en) 2000-12-13 2012-12-18 Nugen Technologies, Inc. Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof
US7771934B2 (en) 2000-12-13 2010-08-10 Nugen Technologies, Inc. Methods and compositions for generation of multiple copies of nucleic acid sequences and methods of detection thereof
US7914997B2 (en) * 2001-01-25 2011-03-29 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US7846668B2 (en) 2001-01-25 2010-12-07 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US7960537B2 (en) 2001-01-25 2011-06-14 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US20050089851A1 (en) * 2001-01-25 2005-04-28 Petr Pancoska Polynucleotides for use as tags and tag complements, manufacture and use thereof
US7943322B2 (en) * 2001-01-25 2011-05-17 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US8624014B2 (en) 2001-01-25 2014-01-07 Luminex Molecular Diagnostics, Inc. Families of non-cross-hybridizing polynucleotides for use as tags and tag complements, manufacture and use thereof
US7927808B2 (en) * 2001-01-25 2011-04-19 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US7927809B2 (en) * 2001-01-25 2011-04-19 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US7608398B2 (en) 2001-01-25 2009-10-27 Luminex Molecular Diagnostics, Inc. Polynucleotides for use tags and tag complements, manufacture and use thereof
US20110039726A1 (en) * 2001-01-25 2011-02-17 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US7645868B2 (en) 2001-01-25 2010-01-12 Luminex Molecular Diagnostics, Inc. Families of non-cross-hybridizing polynucleotides for use as tags and tag complements, manufacture and use thereof
US20100311957A1 (en) * 2001-01-25 2010-12-09 Luminex Molecular Diagnostics, Inc. Families of Non-Cross-Hybridizing Polynucleotides for Use as Tags and Tag Complements, Manufacture and Use Thereof
US20100112705A1 (en) * 2001-01-25 2010-05-06 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US20080014587A1 (en) * 2001-01-25 2008-01-17 Petr Pancoska Polynucleotides for Use as Tags and Tag Complements, Manufacture and Use Thereof
US7846670B2 (en) 2001-01-25 2010-12-07 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US20100216133A1 (en) * 2001-01-25 2010-08-26 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US20100216134A1 (en) * 2001-01-25 2010-08-26 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US20100216135A1 (en) * 2001-01-25 2010-08-26 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US20070244310A1 (en) * 2001-01-25 2007-10-18 Petr Pancoska Polynucleotides for use tags and tag complements, manufacture and use thereof
US7846734B2 (en) 2001-01-25 2010-12-07 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US7846669B2 (en) 2001-01-25 2010-12-07 Luminex Molecular Diagnostics, Inc. Polynucleotides for use as tags and tag complements, manufacture and use thereof
US20030087251A1 (en) * 2001-03-09 2003-05-08 Nurith Kurn Methods and compositions for amplification of RNA sequences
US7771946B2 (en) 2001-03-09 2010-08-10 Nugen Technologies, Inc. Methods, kits and compositions for single primer linear isothermal amplification of nucleic acid sequences
US8071311B2 (en) 2001-03-09 2011-12-06 Nugen Technologies, Inc. Methods and compositions for amplification of RNA sequences
US7354717B2 (en) 2001-03-09 2008-04-08 Nugen Technologies, Inc. Methods and kits for amplification of RNA sequences using composite primers
US7351557B2 (en) 2001-03-09 2008-04-01 Nugen Technologies, Inc. Amplification of RNA sequences using composite RNA-DNA primers and strand displacement
US6946251B2 (en) 2001-03-09 2005-09-20 Nugen Technologies, Inc. Methods and compositions for amplification of RNA sequences using RNA-DNA composite primers
US9181582B2 (en) 2001-03-09 2015-11-10 Nugen Technologies, Inc. Compositions for amplification of RNA sequences using composite primers
US7094536B2 (en) 2001-03-09 2006-08-22 Nugen Technologies, Inc. Methods and compositions for amplification of RNA sequences
US20060014182A1 (en) * 2001-03-09 2006-01-19 Nugen Technologies, Inc. Methods and compositions for amplification of RNA sequences
US20020164628A1 (en) * 2001-03-09 2002-11-07 Nurith Kurn Methods and compositions for amplification of RNA sequences
US9920380B2 (en) 2001-07-03 2018-03-20 The Institute For Systems Biology Methods for detection and quantification of analytes in complex mixtures
US9175325B2 (en) 2003-04-14 2015-11-03 Nugen Technologies, Inc. Global amplification using a randomly primed composite primer
US20050019793A1 (en) * 2003-04-14 2005-01-27 Nurith Kurn Global amplification using a randomly primed composite primer
US7402386B2 (en) 2003-04-14 2008-07-22 Nugen Technologies, Inc. Global amplification using random priming by a composite primer
US8465950B2 (en) 2003-04-14 2013-06-18 Nugen Technologies, Inc. Global amplification using a randomly primed composite primer
US20050026200A1 (en) * 2003-07-28 2005-02-03 Holden David P. Method for error detection and increased confidence of sample decoding
US8912130B2 (en) 2003-08-29 2014-12-16 Illumina, Inc. Methods of forming and using a solid-phase support
US20050048667A1 (en) * 2003-08-29 2005-03-03 Brett Ellman Method of forming and using solid-phase support
US8022013B2 (en) 2003-08-29 2011-09-20 Illumina, Inc. Method of forming and using solid-phase support
WO2005117514A3 (en) * 2004-06-01 2007-05-18 Tm Bioscience Corp Method of detecting cystic fibrosis associated mutations
US20080138803A1 (en) * 2004-06-01 2008-06-12 Galvan-Goldman Nee Galvan Barb Method of Detecting Cystic Fibrosis Associated Mutations
US9588051B2 (en) 2004-09-17 2017-03-07 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US20080161195A1 (en) * 2004-09-17 2008-07-03 Stephen Turner Arrays of optical confinements and uses thereof
US8709725B2 (en) 2004-09-17 2014-04-29 Pacific Biosciences Of California, Inc. Arrays of optical confinements and uses thereof
US9709503B2 (en) 2004-09-17 2017-07-18 Pacific Biosciences Of California, Inc. Apparatus and method for performing nucleic acid analysis
US20080161194A1 (en) * 2004-09-17 2008-07-03 Stephen Turner Arrays of optical confinements and uses thereof
US8431691B2 (en) 2005-02-01 2013-04-30 Applied Biosystems Llc Reagents, methods, and libraries for bead-based sequencing
US9493830B2 (en) 2005-02-01 2016-11-15 Applied Biosystems, Llc Reagents, methods, and libraries for bead-based sequencing
US8329404B2 (en) 2005-02-01 2012-12-11 Applied Biosystems Llc Reagents, methods, and libraries for bead-based sequencing
US20090181860A1 (en) * 2005-02-01 2009-07-16 Applied Biosystems Inc. Reagents, methods, and libraries for bead-based sequencing
US10323277B2 (en) 2005-02-01 2019-06-18 Applied Biosystems, Llc Reagents, methods, and libraries for bead-based sequencing
US9217177B2 (en) 2005-02-01 2015-12-22 Applied Biosystems, Llc Methods for bead-based sequencing
US20100297626A1 (en) * 2005-02-01 2010-11-25 Life Technologies Corporation Reagents, Methods, and Libraries for Bead-Based Sequencing
US20110077169A1 (en) * 2005-02-01 2011-03-31 Life Technologies Corporation Reagents, Methods, and Libraries for Bead-Based Sequencing
EP2316977A1 (en) * 2005-02-01 2011-05-04 AB Advanced Genetic Analysis Corporation Reagents, methods and libraries for bead-based amflication
US8796186B2 (en) 2005-04-06 2014-08-05 Affymetrix, Inc. System and method for processing large number of biological microarrays
US7939258B2 (en) 2005-09-07 2011-05-10 Nugen Technologies, Inc. Nucleic acid amplification procedure using RNA and DNA composite primers
US8852867B2 (en) 2005-09-07 2014-10-07 Nugen Technologies, Inc. Nucleic acid amplification procedure using RNA and DNA composite primers
US9890419B2 (en) 2005-12-23 2018-02-13 Nanostring Technologies, Inc. Nanoreporters and methods of manufacturing and use thereof
US20090062129A1 (en) * 2006-04-19 2009-03-05 Agencourt Personal Genomics, Inc. Reagents, methods, and libraries for gel-free bead-based sequencing
EP4170042A1 (en) 2006-06-14 2023-04-26 Verinata Health, Inc. Methods for the diagnosis of fetal abnormalities
EP2366801A1 (en) 2006-06-14 2011-09-21 Verinata Health, Inc Methods for the diagnosis of fetal abnormalities
EP2589668A1 (en) 2006-06-14 2013-05-08 Verinata Health, Inc Rare cell analysis using sample splitting and DNA tags
EP3406736A1 (en) 2006-06-14 2018-11-28 Verinata Health, Inc Methods for the diagnosis of fetal abnormalities
EP4108780A1 (en) 2006-06-14 2022-12-28 Verinata Health, Inc. Rare cell analysis using sample splitting and dna tags
EP3424598A1 (en) 2006-06-14 2019-01-09 Verinata Health, Inc Rare cell analysis using sample splitting and dna tags
US20090191553A1 (en) * 2007-10-01 2009-07-30 Applied Biosystems Inc. Chase Ligation Sequencing
US9334530B2 (en) 2007-11-06 2016-05-10 Ambergen, Inc. Methods for making and imaging arrays that comprise a plurality of different biomolecules
US20090286286A1 (en) * 2007-11-06 2009-11-19 Ambergen , Inc. Methods for controlling amplification
US20100256015A1 (en) * 2007-11-06 2010-10-07 Ambergen, Inc. Methods For Making And Imaging Arrays
US20090181390A1 (en) * 2008-01-11 2009-07-16 Signosis, Inc. A California Corporation High throughput detection of micrornas and use for disease diagnosis
US8034568B2 (en) 2008-02-12 2011-10-11 Nugen Technologies, Inc. Isothermal nucleic acid amplification methods and compositions
US7846666B2 (en) 2008-03-21 2010-12-07 Nugen Technologies, Inc. Methods of RNA amplification in the presence of DNA
US11705217B2 (en) 2008-03-28 2023-07-18 Pacific Biosciences Of California, Inc. Sequencing using concatemers of copies of sense and antisense strands
US10077466B2 (en) 2008-08-14 2018-09-18 Nanostring Technologies, Inc. Stable nanoreporters
US9856519B2 (en) 2008-08-14 2018-01-02 Nanostring Technologies, Inc. Stable nanoreporters
US9416407B2 (en) 2008-10-01 2016-08-16 Agilent Technologies, Inc. Method for genome analysis
US20100081576A1 (en) * 2008-10-01 2010-04-01 Ach Robert A Method for genome analysis
US9995739B2 (en) 2009-10-13 2018-06-12 Nanostring Technologies, Inc. Protein detection via nanoreporters
US9714937B2 (en) 2009-10-13 2017-07-25 Nanostring Technologies, Inc. Protein detection via nanoreporters
US11390918B2 (en) 2009-11-06 2022-07-19 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US10988804B2 (en) 2009-11-06 2021-04-27 The Board Of Trustees Of The Leland Stanford Junior University Nucleic acid sequencing apparatus for monitoring status of a transplant recipient
US10968479B2 (en) 2009-11-06 2021-04-06 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US10982275B2 (en) 2009-11-06 2021-04-20 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US11384389B2 (en) 2009-11-06 2022-07-12 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US10329607B2 (en) 2009-11-06 2019-06-25 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US9845497B2 (en) 2009-11-06 2017-12-19 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US11098350B2 (en) 2009-11-06 2021-08-24 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US10494669B2 (en) 2009-11-06 2019-12-03 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
WO2011057061A1 (en) 2009-11-06 2011-05-12 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US8703652B2 (en) 2009-11-06 2014-04-22 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US11597966B2 (en) 2009-11-06 2023-03-07 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
EP3719140A1 (en) 2009-11-06 2020-10-07 The Board Of Trustees Of The Leland Stanford Junior University Non-invasive diagnosis of graft rejection in organ transplant patients
US9714446B2 (en) 2010-02-11 2017-07-25 Nanostring Technologies, Inc. Compositions and methods for the detection of small RNAs
US9758834B2 (en) 2011-03-28 2017-09-12 Nanostring Technologies, Inc. Compositions and methods for diagnosing cancer
WO2013043922A1 (en) 2011-09-22 2013-03-28 ImmuMetrix, LLC Compositions and methods for analyzing heterogeneous samples
WO2013085710A2 (en) 2011-12-09 2013-06-13 Illumina, Inc. Expanded radix for polymeric tags
EP2824180A4 (en) * 2012-03-05 2016-03-02 Ngk Insulators Ltd Method for detecting target nucleic acid
US10722858B2 (en) 2013-03-15 2020-07-28 Lineage Biosciences, Inc. Methods and compositions for tagging and analyzing samples
US11161087B2 (en) 2013-03-15 2021-11-02 Lineage Biosciences, Inc. Methods and compositions for tagging and analyzing samples
US10640816B2 (en) 2015-07-17 2020-05-05 Nanostring Technologies, Inc. Simultaneous quantification of gene expression in a user-defined region of a cross-sectioned tissue
US10501777B2 (en) 2015-07-17 2019-12-10 Nanostring Technologies, Inc. Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue
US11708602B2 (en) 2015-07-17 2023-07-25 Nanostring Technologies, Inc. Simultaneous quantification of gene expression in a user-defined region of a cross-sectioned tissue
US10415080B2 (en) 2016-11-21 2019-09-17 Nanostring Technologies, Inc. Chemical compositions and methods of using same
US11279969B2 (en) 2016-11-21 2022-03-22 Nanostring Technologies, Inc. Chemical compositions and methods of using same
US11821026B2 (en) 2016-11-21 2023-11-21 Nanostring Technologies, Inc. Chemical compositions and methods of using same
CN110050071A (en) * 2016-12-09 2019-07-23 乌尔蒂维尤股份有限公司 The improved method that nucleic acid preparation for using label carries out multiplexing imaging
WO2018107054A1 (en) * 2016-12-09 2018-06-14 Ultivue, Inc. Improved methods for multiplex imaging using labeled nucleic acid imaging agents
US11754562B2 (en) 2016-12-09 2023-09-12 Ultivue, Inc. Methods for multiplex imaging using labeled nucleic acid imaging agents
US11473142B2 (en) 2018-02-12 2022-10-18 Nanostring Technologies, Inc. Chemical compositions and uses thereof
US11377689B2 (en) 2018-02-12 2022-07-05 Nanostring Technologies, Inc. Chemical compositions and uses thereof
US11549139B2 (en) 2018-05-14 2023-01-10 Nanostring Technologies, Inc. Chemical compositions and methods of using same
WO2019229792A1 (en) * 2018-05-28 2019-12-05 株式会社ニコン Method for detecting target nucleic acid fragment

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