CA1317207C - Solution phase nucleic acid sandwich assay and polynucleotide probes useful therein - Google Patents

Solution phase nucleic acid sandwich assay and polynucleotide probes useful therein

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Publication number
CA1317207C
CA1317207C CA000616159A CA616159A CA1317207C CA 1317207 C CA1317207 C CA 1317207C CA 000616159 A CA000616159 A CA 000616159A CA 616159 A CA616159 A CA 616159A CA 1317207 C CA1317207 C CA 1317207C
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nucleic acid
complementary
sequence
analyte
label
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French (fr)
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Michael Steven Urdea
Brian Warner
Thomas Horn
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Bayer Corp
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Chiron Corp
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06Pyrimidine radicals
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06Pyrimidine radicals
    • C07H19/10Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • 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
    • C12Q1/6816Hybridisation assays characterised by the detection means
    • C12Q1/682Signal amplification
    • 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
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701Specific hybridization probes
    • C12Q1/706Specific hybridization probes for hepatitis
    • 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/50Improvements relating to the production of bulk chemicals
    • Y02P20/55Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
    • 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
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/81Packaged device or kit

Abstract

ABSTRACT OF THE DISCLOSURE
Methods and compositions are provided for rapid detection of nucleic acid sequences. The method employs two reagent sets. The first set is a labeling set comprising: (1) a first nucleic acid sequence probe having an analyte complementary region and a first recognition sequence region and (2) a labeled sequence complementary to the first recognition sequence region.
The second set is a capturing set comprising: (1) a second nucleic acid sequence probe having an analyte complementary region and a second recognition sequence region, (2) a specific binding pair member conjugated to a sequence complementary to the second recognition sequence, and (3) a separating means to which is bound a complementary specific binding pair member. The sample and probes are combined under annealing conditions, followed by addition of the other reagents, separation of the bound label from the supernatant and detection of the label in either phase. The invention also encompasses nucleic acid probes formed from one or more modified, derivatizable nucleotides.

Description

SOLUTION PHASE NUCLEIC ACID S~NDWICH ~SSAY
AND POLYNUCLEOTIDE PROBES USEFUL THEREIN

DescriPtion Technical Field This invention relates generally to a solution phase nucleic acid sandwich assay and polynucleotide probes useful therein. The invention also relates to labeled, modified nucleotides which are incorporated in the probes.

Description of Relevant Literature Meinkoth and Wahl, Anal. Biochem., (19B4) 138-267-284, provide a eeview article of hybridiza~ion techniques. See also ~eary et al., Proc. Natl. Acad.
Sci. US~ (1983) 80:~045-4049, for a description of the dot blot assay. Sandwich hybridization i8 described by Ranki et al., Cure. ToP. Microbiol. Immunoloqv (1933) pp. 308ff. See also Ranki et al., Gene (1983) 21:77-85, Virtanen et al., Lancet (1983) 381-383, and U.S. Patent No. 4,486,539. EPA 123,300 describes biotin-avidin complexes ~or use in detecting nucleic acid sequences.
Sung, in Nucl. Acids Res. 9(22):6139-6151 ~1981) and in J. Orq. Chem. 47:3623-3628 (1982), discusses ~he synthesis of a modified nucleotide and application of the modified structure in oligonucleo~ide synthesis.
Modified nucleotides are ~al80 discussed in braper i ) -2~ 1317207 Nucleic Acids Res. lZ:2:989-1002 (1984), whecein it is suggested that cytidine residues in RNA be modified so as to bind to repor~er molecules. Later work suggests similar modification o cytidine residues in DNA (Anal.
Che~. 157(Z):l99 (1986). European Patent Application 06387g, filed 6 April 1982, and PCT Application No.
PCT/US84/00279 also describe modified nucleotides and applications thereof.

Background Art The increasing ease of cloning and synthesizing DNA sequences has greatly expanded opportunities for detecting particular nucleic acid sequences of interest. No longer must one rely on the use of immunocomplexes for the detection of pathogens, lesions, antigens, and the like. Rather than detecting particular determinant sites, one can detect DNA
sequences or RNA sequences associated with a particular cell. In this manner, diseases can be diagnosed, phenotypes and genotypes can be analyzed, as well as polymorphisms, relationships between cells, and the like.
For the most part, analyses of DNA sequences have involved the binding of a sequence to a solid support and hyhridization o~ a complementary sequence to the bound sequence. The annealing and complexing step usually involves an extended period of time and requires careful washing to minimize non-specific background signals. There is substantial interest in developing new techniques for analyzing nucleic acid seguences, which are more rapid, minimize the number of manipulative steps, and provide for an increased signal to noise ratio.
.

1 ~ ~
1 31 72~7 This application is also directed to polynucleotide erobes useful in 6uch techniques. The majority of polynucleotide probes in current u~e are radioactively labeled, e.g. with isotopes of hydrogen ( H), phosphorus ( P), carbon ( C) or iodine ( I). These materials are relatively si~ple to syn~hesize by direct inclusion of the radioactive moieties, e.g. by kinasing with P-labeled ATP, equilibrating with tritiated water, or the like. As is well known, however, use of such radioac~ive labels has drawbacks, and other detectable species which are not radioactive are preferred.
In order to incorporate other, non-radioactive types of detectable species in a nucleotide, some sort of chemical modification of the nucleotide is required.
It is widely recognized that nucleotide modification is a difficult and sensitive procedure, as any modification reaction has to be mild enou~h to leave the RNA or DNA
molecules intact, while giving a modified nucleotide product which can participate in normal base pairing and stacking interactions. These considerations typically limit nucleotide substitution positions to the 5-position of a pyrimidine and the 8-position o~ a purine, as noted in the literature (see, e.g., European Patent Application 063879, cited supra).
Other considerations ~ust also be taken into accoun~. Base pairing may be hindered durinq hybridiza~ion if the detectable label is at one end of the nucleotide chain rather than presen~ at some point within it. Further, it has proved difficult to provide even non-radioactively labeled probes which may be inexpensively synthesized in large quantity. Thus, many known prDbes are~limited in their potential applications.

, ~4~ 1 31 7 2 07 Disclosuce of the Invention Methods and compositions are provided for detecting particular nucleic acid seguences. Two sets of reagents are employed, which are referred to as the capturing set and the labeling set. Each set has at least two members. The labeling se~ has (13 a first probe set, which comprises one or a group of first analyte complementary sequence-first label reagent recognition sequence conjugate(s); and (2) one or a group of sequences complementary to said first recognition sequence-label conjugate(s). The captuLing set has (1) a second probe set, which comprises one or a group of second analyte complementary sequence(s3 joined to second capturing reagent polynucleotide recognition sequence(s); (2) one or a group of sequences complementary to said second capturing recognition sequence(s) bound to a separation member or preferably a f irst specific binding pair member to define the capturing conjugate; and (3) a separation membe~ joined zo to a first complementary specific binding pair member when ~2) does not have the separation member.
The single stranded nucleic acid sample may be joined with the probes containing the complementary sequences o the two sets under annealing conditions, followed by the addition of the cap~uring and optionally ~he labeling conjugates to provide for the analyte complex with the specific binding pair member and optionally the label. The probe hybridized analyte sequence is separated by combining the complex with the separating means and separating probe bound analyte from unbound analyte. Where the label has not been previously added, the-first recognition sequence-label conjugate is added to the phase containing the ~ _5_ I 31 7207 separation member under hybridizing conditions, The label may then be detected in either phase.
.In another aspect of ~he invention, a modified, derivatizable nucleotide is pr~vided having the structure of Formula 1:

RZ

(IH2)x NH
1 ~ R3 ~ N

R0 - , \/ Y

~
ORs R6 Formula 1 wherein R is a reactive group derivatizable with a detectable label, which reactive yroup may be amine, carboxyl or thiol and further may be protec~ed for various synthetic manipulationsO R is an o~tional linking ~oiety such.as those typically used to label proteins, and includes an amide, thioether or di~ulfide linkage or a combination the~eof, R is selected from the group consisting of hydrogen, methyl, bromine, fluorine and iodine, R is hydrogen, an anchoring ' ~
-6- 1317~07 group which covalently binds the structure to a solid support, or a blocking group such as dimethoxytrityl or pixyl, which blocking group is generally base-stable and acid-sensitive, R5 is hydrogen, an anchoring group ~ 5 which covalen~}y binds the ~tructu~e to a solid support, - or a phosphorus derivative enabling addition of nucleotides at the 3' position, and may be, for example, PO3H2, a ~hosphotriester, a phosphodiester, a phosphite, a phosphoramidite, H phosphonate or a 10 phosphorothioate, and R is H, OH, or OR where R is a functional group useful as a protecting moiety in RN~
synthesi6, and x is an integer in the range of l and 8 inclusive. The invention also encompasses a method of making the above modified nucleotide including the step 15 of derivatizing the R moiety with a detectable label.
In still another aspect, nucleic acid probes are provided using one or more of the above modified nucleotides. The probe can be used to screen a sample containing a plurality of single-stranded or 20 double-stranded polynucleotide chains, and will label the desired se~uence, if present, by hybridization.

Brief DescriPtion of the Drawinqs Figure l is an illustrative depiction of a 25 complex from the various com~onents bound to a solid support (1? using DNA bridges for non-covalent binding and (2) using biotin-avidin bridges for non~covalent binding.

30 Modes for Carryinq Out the Invention l. Sandwich AgsaY Method Methods and compositions are ~rovided for detecting a nucleic acid sequence by empl~ying two sets -7_ 1 3 i 7~07 of reagents. By using combinations of nucleic acid sequences complementary to a nucleic acid analyte and ~o arbitrar~ sequences and specific binding pair members, a detectable label may be separated into two phases in proportion to the amount of analyte present in a sample. By providing for annealing of nucleic acid sequences in solution, the time for performing the assay can be substantially diminished as compared to annealing on a solid surface and the number of separations and washing steps requiced can be limited and be less critical, so as to reduce technician error. Reagents containing complementary sequences can be added in excess during or at the end of the denaturation to inhibit renaturation of double stranded DNA and to react rapidly with the analy~e strand by diffusion in solution. The rate of binding ~o the solid suppor~ can also be accelerated by the presence of a large amount of the binding pair member bound to the su~port. In addition, by adding the label conjugate as the last ZO reagent, the analyte will be present in a highly concentrated ~orm.
As indicated above, the method involves two sets of reagents. The first set results in labeling the analyte sequence. The second set provides the means for separating label bound to analyte from unbound label in the assay medium.
The first set, the labeling set, will involve at least two reagents and may involve 10 to 30 reagents or mo~e. The first reagent will be a subset of nucleic acid reagents and each member of the subset will have two nucleic acid regions. The first nucleic acid region - of each me~ber of the subset will be a region - complementary to a sequence of the analyte. The second - nucleotide sequence will be a recognition site or the -~- 1 31 7207 labeling reagent. This second sequence will be selected, so as not to be encoun~ered by endogenous sequences in the sa~ple.
The subsets will have regions complementary to ~ 5 the analyte sequence of at least 15 nucleotides (nt), usually at least 25nt, mor~ uually at least 50nt, and not more than about 5kb, usually not more than about lkb, ~referably not more than about lOOnt. The sequence complementary to the analyte may be joined to a non-specific sequence at ~ither or both the 5l and 3'-termini. The non-complementary sequence, if judiciously selected so as not ~o bind to sequences in the assay which could result in false positives, can be of any length, usually fewer than lOkb, more usually fewer than 5kb.
The complementary sequences will be chosen so as to leave areas for binding of the other reagents to ; the analyte. Usually, areas of at least ~5nt will be left available, where the analyte sequences complementary to the sequences of the individual members of the reagent subse~ may be substantially contiguous or separated and members of one subset may alternate with members of the other sub~et. The particular pattern of binding between the two subsets may vacy widely depending on the sequences of the analyte.
The reagent sequences may be prepared by ~ynthesis in accordance with conventional procedures or by cloning and may be modified as appropria~e for labeling.
The set of sequences which are complemen~ary to the analyte may be selected based on a variety of considerations. Depending upon the nature of the analyte, one may be interested in a consensus sequence, a sequence associated with polymorphisms, a particular , g 13172~7 phenotype or genotype, a particular strain, or the like. Thus, the labeling complementary sequences will be chosen in conjunc~ion with the other complementary sequences of the capturing set ~o provide information -5 concerning the analyte.
-The labeled sequence will include a fiequence complementary to the first recognition sequence of the labeling probe(s). The labeling sequence will include one or more molecules, which directly or indirectly provide for a detectable signal. The labels may be bound to individual members of the complementary sequence or may be present as a terminal member or terminal tail having a plurality of labels. Various means for providing labels bound to the sequence have been repor~ed in the literature. See, for example, Leary et al., Proc. Natl. Acad. Sci. USA (1983) 80:4045;
Renz and Kurz, Nucl. Acids Res. (1984) 12:3435:
Richardson and Gumport, ~ucl. Acids Res. (lg~3) 11:6167;
Smith et al., Nucl. Acids Res. (1985) 13:2399; Meinkoth and Wahl, Anal. Biochem. (1984) 138:267. The labels may be bound either covalently or non-covalently to the complementary sequence.
Labels which may be employed include radionuclides, fluorescers, chemiluminescers, dyes, enzymes, enzyme substrates, en~yme cofactors, enzyme inhibitors, enzyme subunits, metal ions, and the like.
Illustrative specific labels include fluorescein, rhodamine, Texas red, phycoerythrin, umbelliferone, luminol, NADPH, a-B-galactosidase, horsaradish peroxidase, etc.
The labeled sequence can be conveniently -prepared by synthesis~ By providing for a terminal group which has a convenient functionality, various - - labels may be joined thcough the functionality. Thus, -lO- 1~17207 one can provide ~or a carboxy, thiol, amine, hydrazine or other functionality to which the various labels may be ioined without detrimentally affecting duplex formation with the sequence. As already indicated, one can ha~e a molecule with a pIurality of labels joined to the sequence complementary to the labeling ~iequence.
~lternatively, one may have a ligand bound to the labeling sequence and use a labeled recep~or for binding to the ligand to provide the labeled analyte complex.
The second set of reagentsi provides the means for separation of label bound to analyte from unbound label. The means for ~he sepatation or capturing means involves at least one capturing probe, usually a plurality of probes defining a subset, which includes two polynucleotide sequence regions that include a second subset of sequences complementary to the analyte, differing from the first subset of complementary sequences of the labeling probe and a recognition sequence, different from the first subset recognition sequence of the labeling probe. The second set of recognition sites for the capture probes may lie between the first set of recognition sites for the labeling probes as described above. The capturing sequences will be selected and synthesized in the same manner as described above using the considerations dicecting the selection for the labeling probes. Thus, the same constraints will be involved in preparing the cap~uring probes.
While the separating means may be directly bound to a ~equencs complementary to the capturing recognition sequence, preferably a specific binding pair member will be bound to the complemen~ary isequence. The specific binding pair! member will be a ligand or receptor, preferably a ligand. Ligands may be any - 1317~07 molecules for which a naturally occurring receptor exists or can be prepared. Thus, naturally occurring lig~nds may be exemplified by biotin, thyroxine, enzyme substrates, ~teroids, and the like. In~tead of 5 naturally occurring liqands, any hapten may be employed for the eroduction of antibodies. Ligands will generally be at least about 125 molecular weight and usually less than about 5,000 molecular weight, moce usually less than about 2,000 molecular weight, and preferably le~s than about 1,000 molecular weight.
The recep~ors will generally be protein molecules and may include antibodies, naturally occurring proteins, such as avidin, thyroxine binding globulin, etc., lectins, enzymes, and the like. The receptors will generally be at least about 10,000 molecular weight, more usually 12,000 or more molecular weight, usually less than about one million molecular weight.
The specific binding pair member may be joined to the second recognition sequence by any convenient means. As already indicated, the sequence may be synthesized, providing for a convenient functionality at the terminal base, which may then be used a6 the linkage site. One or a plurality of specific binding pair members may be joined to the complementary sequence, depending upon the particular choice of the specific binding pair member, its 6ize, and the nature of the functionalities. ~lternatively, for a large specific binding pair member, a plurality o sequences may be joined to the binding pair member. The caeturing conjugate will be prepared, so that there will be little interfeLence, if any, from ~he specific binding pair member with the annealing of the complementary .

-lZ- ~317201 recognition sequences and from duplex formation with the ligand-receptor binding.
. .Alternatively, the receptor may be an additional nucleotide sequence that specifically recognize~ the recognition sequence of the cap~uring probe.
The ~eparation means can be any support which allows for a rapid and clean separation of label bound to analyte fro~ unbound label. Thus, the separation means may be particles, a solid wall surfaca of any of a variety of containers, e.g., centrifugal tubes, columns, microtiter plate wells, filters, tubing, etc.
Preferably, particles will be employed of a size in the range of about 0.4 to 200~, more usually from about 0.8 to 4.0~. The particles may be any convenient material, such as latex, glass, etc.
The homologous nucleic acid sequences need not have perfect complementarity to provide homoduplexes.
In many situations, heteroduplexe~ will sufice where fewer than 15%, usually fewer than 10% of the bases are mismatches, ignoring loops of f ive or more members.
Samples of analyte nucleic acids may be from a variety of sources, e.g., biological fluids or solids, food stuffs, environmental materials, etc., and may be prepared for the hybridization analysis by a variety of means, e.g., proteinase K/S~S, chaotropic salts, etc.
Also, it may be of advantage to decrease the aveLage size of the analyte nucleic acids by enzymatic, PhYsical or chemical ~eans, e.g., restrictlon enzymes, fionication, chemical degradation (e.g., metal ions), etc. The fragmen~s m~y he as small as O.}kb, usually being at least about 0.5kb and may be lkb or higher.
In caLrying out the method, the analyte sequence wili be provided in single s~randed form.

.

Where the ~equence i5 naturally present in single stranded form, denaturation will not be required.
However, where the sequence is present in double stranded form, the sequence will be denatured.
Denaturation can be carried out by various techniques, such as alkali, generally from about O.OS to 0.2M
hydroxide, formamide, detergents, heat, or combinations thereof. Denaturation ~an be carried out in the presence of the labeling probe and/or the aapturing probe, so that upon change of conditions to annealing conditions, the probes will bind to any co~plementary sequences which are present. For example, where heat and alkali are employed, by neutralization and cooling, annealing will occur.
In many ~ituations, it will be preferable to avoid having either the label or the seearation means present during denaturation. The elevated temperatures, the non-aqueous solvents, the salts, or other materials present during denaturation may result in degradation, or undesirable modification of the label ~nd/or separation means. Therefore, in many situations, denaturation may occur in the presence of the probes, whereupon cooling rapid annealing of the p~obes to the single-stranded DNA may occur, followed by the addition of the other reagents at lower temperatures and, as appropriate, under milder conditions, such as neutral pH, ~educed ionic strength, or the like.
Normally, the ratio of probe to anticipated moles of analyte will be at least 1:1, pre~erably at ~0 least abou~ 1.5:1, and more preferably 2:1 and may be as high as 100:1 or higher. Concentrations of each of the - probes will generally ~ange from about 10 to M, with sample nucleic:ac}d concentrations varying from 10 21 to 10-12M;

After annealing conditions have been achieved, or even prior to such time, the labeled ~irst recQgnition sequence and the capturing second recognition sequence are added and allowed to hybridize. Alternatively, the labeled first recognition sequence can be added after capture and separa~ion.
A preferred embodiment which greatly reduces background and provides for extraordinarily high sensitivity will employ the ~ollowing sequence. With double-stranded analyte, the analyte will be denatured in the presence of the erobe or complementary sequences, or the probes may be added shortly after denaturation, and under annealing conditions. After sufficient time for annealing, the complexes may then be combined with ~he separation means, whereby the complexes will be bound to the support. Any background DNA OL
non-seecifically bound DNA may be washed away so as to avoid non-specific binding of label in the next step.
The solid support may then be washed to remove any non-specifically bound label to provide for a substantially reduced background of non-specifically bound label.
Consider Figure 1, part 2. In effect, the analyte which i6 the long bar at the top is combined with the A and B probes, where A provides the complementary sequence for the label conjugate and B
pro~ides the complementary sequence for the specific binding pair member, in this case, biotin. Thu~, the A
and B probes and the analyte would be joined together under annealing conditions, whereby complex formation would occur between the probes and the analyte. The biotin conjugate, B' could be included with the probes or be added in a separate ,step to the solution containing the analyte complexes~ Af~er sufficient time for B' to anneal to B, the resulting biotinylated analyte complex would then be added to the solid ~upport to which avidin i6 bound. After sufficient time for the ~pecific binding pair members to form complexes, the solid support could be washed free of any non-specific D~A, followed by the addition of the labeled sequence, - which in this case is indicated as being fluorescein bound to A~. ~he labeled sequence would be added under annealing conditions and after sufficient time for duplex formation, non-specifically bound and excess labeled conjugate would be washed away and the fluorescence of the surface determined.
A somewhat shortec protocol is provided by the configuration depicted in part 1 of Figure 1. In this situation, the peobes A and B would be added to the analyte under annealing conditions, whereby analyte complexes would form. After sufficient time for analyte complexes to form, the analyte complex solution would then be added to ~he solid support for sufficient time for the capturing probes to bind to the solid support by complex formation with the sequence indicated as B'C.
Excess DNA could be washed away, followed by the addition of the fluorescein labeled sequence ~', and the mixture allowed to anneal for su~ficient time for complex formatiorl to occur between the label and the probes. Excess in non-specifically bound label could then be washed away to provide the configuration depicted in Figure 1, part 1.
Usually, the dena~uring step will take from about 5 to 25 minutes, usually from about 5 ~o 15 minutes, while the annealing s~ep will generally take - from about 30 minutes to 2 hours, frequently being completed in about 1 hour. -Annealing can be carried vut at a mildly elevated tem~erature, generally in the range -16- 13172~)7 from abou~ 20C to 50C, more usually from about 25C ~o 40C, particularly 37C.
~ U~ually, an aqueous mediu~ is employed, particularly a buffered aqueous medium, which may include various additive~. Additives which may be employed include low concentrations of detergent (0.1 to 1%, salts, e.g., sodium citrate (0.017 to 0.170M), Ficoll, polyvinylpyrrolidone, carrier nucleic acids, carrier ~roteins, etc. Depending u~on the nature of the specific binding pair members, variou6 solvents may be added to the aqueou~ medium, such as dimethylform amide, dimethylsulfoxide, and formamide. These other solvents will be present in amount6 ranging from 2 to 50%.
The stringency of the annealinq medium may be controlled by temperature, salt concentration, solvent system, and the like. Thus, depending upon the length and nature of the sequence of interest, the stringency will be varied.
For the separa~ion step, for example, using a ligand-receptor pair, the medium may be changed to optimize or approximately optimize the conditions for specific binding pair complex formation. Thus, the p~
will usually be modified to be in the range oE about 6 to 9, preferably about 7. This can be readily achieved, by adding from about 0.5 to 2, usually about 1 volume of about a 0.1 to 0.5M buffered medium, e.g., phosphate buffered saline, to the annealing medium. This medium may be added in conjunction with the separation means and the mixture allowed to incubate for at least Smin., usually about lOmin., and less than about 60min., usually about 15 to 45min., mo~e usually abou~ 30min.
being satisfactory.
The ~hases may then be se~arated in accordance with the nature of the separation means. For particles, centrifugation or filtration will provide for separation of the particles, discarding the supernatant or isolating the supernatant. ~here the particles are assayed, the particles will be wa~hed thoroughly, usually from one to five times, with an appropriate buffered medium, e.g., PBS. When the separation means is a wall or ~upport, the supernatant may be isolated or discarded and the wall washed in the same manner as indicated for the particle~.
Depending upon the nature of the label, various ~echniques can be employed for de~ecting the presence of the label. For fluorescers, a large number of different fluorometers are available. With enzymes, either a fluorescent or a colored product can be provided and determined fluorometrically, spectrophoto metrically or visually. The various labels which have been employed in immunoassays and the technigues applicable to immunoassays can be employed with the subject as~ays.

2. Nucleic Acid Probes Nucleic acid probes useful in conjunction with ~he above assay method are probes which are prepared from one or more modified nucleotides. As used herein, the following definitions apply:
"Derivatizable" nucleotides are nucleotides modified so as to include at the 4-position of a eyrimidine a functional group which can react with a detectable label. An example of a derivatizable nucleotide is one which has been modified at the 4-position with an alkylamine moiety so ~hat a free amine group is present on the structure.
"Derivatized" nucleotide~ are nucleotides in ! which the derivatizable functional grQUp a~ the .
' ~, ' , .

1 31 72(J7 4-position of the pyrimidine i8 bound, covalently or otherwise, directly or indirectly, to a detectable label.
"Alkylamine nucleotides" are nucleotides having an alkylamine group at the 4-position of a pyrimidine, bound to the structure in such a way as to ~rovide a free amine group at Shat position.
A "polynucleotide" is a nucleotide chain structure containing at least two nucleotidqs. The "polynucleotide erobe" provided herein is a nucleotide chain structure, as above, containing at least two nucleotides, at least one of which includes a modified nucleotide which has substantially the same structure as that given by Formula 1.
"Detectable label" refers to a moiety which accounts for the detectability of a complex or reagent.
In general, the most common types of labels are fluorophores, chromophores, radioactive isotopes, and enzymes.
"Fluorophore" refers to a substance or portion thereof which is capable of exhibi~ing fluorescence in the detectable range. Typically, this fluore~cence is in the visible region, and there are common techniques for its guantitation. Examples of fluorophores which are commonly used include fluorescein ~usually supplied as fluorescein isothiocyanate [FITC] or fluorescein amine), rhodamine, dansyl and umbelliferone.
Formulae 2 through 5 illustrate the nucleotide numbering scheme used herein.

,, - . , ~ ' ' , ~~ 110~`~H~

2a (R=OH) 3a (R=OH) 2b (R=H~ 3b (R=H) HOC~ H~e HO R 1~0 R

4a (R--OH~ 5a (R=OH, Rl=H) 4b (R=H~ Sb (R=H, R'=CH3) In a preferred embodiment, the substituents of the modified nucleotide of Formula 1 are as 0110w6.
R , which is a reactive group derivatizable with a detectable label, is preferably -NH2, -COOH or -SH.
R is an optional linker moiety which contains an amide, thioether or disulfide linkage, or a combination thereof. R is preferably a heterobifunctional linker such as t~hose typically used to bind proteins to labels. In most cases, a free amino group on a prvtein or other structure will react with a carboxylic acid or.activated ester moiety of the unbound R compound so as ~o bind the linker via an amide linkage. Other methods of binding the linkec to the nucleotide are also possible. Examples of particularly preferred linkers include S O _ -NHC(CH2)X- ~ Rl]

Formula 6 o -NHC(CH2)x-Ss-(cH2)x Formula 7 ~ ~(CH~)x ~-R ]
Formula 8 wherein x is an in~eger in the range of 1 and 8 inclusive.
As may be seen in Formula 1, the linker, if present, is attached to ~he nucleotide structure through an alkylamine functionality -~H-(CH2)X- wherein x is an integer in the range of 1 and 8 inclusive, and the alkylamine functionality is present at the 4-position of the pyrimidine base.
As noted above, R is hydrogen, methyl, bromine, fluorine or iodine. Thus, the base of the nucl~otide i6 a pyrimidine optionally substituted at the 5-~osi~ion with the aforementioned R substituents.
R is typically hydrogen, if che modified nucleotide is a terminal 5' structure, or a suitable blocking group useful in polynucleotide synthesi~.
Examples of suitable blocking groups include substituted and unsub6tituted aralkyl compounds, where the aryl i.s, e.g., phenyl, naphthyl, furanyl, biphenyl and the like, and wher.e the substituents are from 0 to 3, usually 0 to 2, and include any non-interfering stable groups, - 5 neutral or polar, electron-donating or withdrawing, generally being of 1 to 10, usually 1 to 6 atoms and generally of from 0 to 7 carbon atoms, and may be an aliphatic, alicyclic, aromatic or heterocyclic groue, generally aliphatically satura~ed, halohydrocarbon, e.g., trifluoro~ethyl, halo, thioetheL, oxyether, ester, amide, nitro, cyano, sulfone, amino, azo, etc.
In one or mole steps ducing nucleotide chain synthesis, it may be desirable to re~lace the hydrogen atom oc blocking group at the R position with a more stable, "capping" group. Suitable capping groups include acyl groups which provide for stable esters.
The acyl groups may be organic or inorganic, including carboxyl, phosphoryl, pyrophosphoLyl, and the like. of particular interest are alkanoic acids, moce particularly aryl-substituted alkanoic acids, where the acid is at least-4 carbon atoms and not more than about 12 carbon atoms, usually not more than about 10 carbon atoms, with the aryl, usually phenyl, substituted alkanoic acids usually of from a ~0 12 carbon atoms.
Various heteroatoms may be present such as oxygen toxY)~
halogen, nitrogen, e.g., cyano, etc. For the most part, the carboxylic acid ester~ will be base labile, while mild acid stable, particularly at moderate tempecatures below about 50C, more particularly, below about 35C
and at pHs greater than about 2, more particularly greater than about 4.
The modif;ed nucleotide may also be attached to a support through the R position so as to facilitate addition of labeled or unlabeled nucleotides at the 3' .

-zz- l3l72n7 (R ) position. In such a case, R is an anchoring group as will be described below. Covalent attachment to a support i8 also preferred during sample screening, as the time and complexity of separating the hybridized S nucleotide chains from ~he ~a~ple is substantially reduced. When the modified nucleotide of Formula 1 is bound to one or more additional nucleotides at the 5' position, the R substituent is eeplaced with such additional nucleotides which are bound through their 3' phosphate groups.
R , as noted, is hydrogen or a phosphorus derivative such as PO3H2, a phosphotriester, a phosphodiester, a phosphite, a phosphoramidite, an H-phosphonate or a phosphorothioate suitable for polynucleotide synthesis, which derivative enables sequential addition of nucleotides at the 3' position.
More generally, such phosphorus derivatives are given ~y Formula 9 and Formula 10:

o oX
~1 /
--O-- 1_o~ _o_p Y

Formula 9 Formula 10 ~5 wherein X is preferably hydrogen or an aliphatic group, particularly a saturated aliphatic group, a ~-heterosubstituted aliphatic group, where the B-substituent is an electron-with~rawing group which readily participates in B-elimination, either as ~he leaving group or ~he proton-activating group, subs~ituted methylene, where the substituent ~ay vary widely and supports a negative charge on the methylene - through inductive or resonating effects; aryl; and aralkyl~ DeRending on the nature of ~he phosphoru6 functionality, one gLOUp may be chosen over another.
Thus, depending upon whether a phosphochloridite, phospho~amidite, phofipha~e, thiophosphate, phosphite, or s the like, is employed, particulàr phosphoro ester groups will be preferred.
Similarly, the groups employed for Y will depend upon the nature of the phosphorus derivative employed for oligomerization~ When the phosphoramidite is employed, Y will have the formula -NT T , where T and T may be the same or different and may be hydrocarbon or have from 0 to 5, usually 0 to 4 heteroatoms, primarily oxygen as oxy, sulfur as thio, or nitrogen as amino, particular tert.-amino, N02 or cyano. The two T's may be taken together to form a mono- or polyheterocyclic ring having a total of from 1 to 3, usually 1 to 2 heteroannular members and from 1 to 3 rings. Usually, the two T's will have a total of from 2 to 20, more usually 2 to 16 carbon atoms, where the T's may be alipha~ic (including alicyclic~, particularly saturated aliphatic, monovalent, or, when taken together, divalent radicals, defining substituted or unsubstituted heterocyclic rings The amines include a wide variety of ~atura~ed secondary amines ~uch as dimethylamine, diethylamine, diisopropylamine, dibutylamine, methylpropylamine, me~hylhexylamine, methylcyclopropylamine, ethylcyclohexylamine, methylbenzylamine, methylcyclohexylmethylamine, butylcyclohexylamine, morpholine, thiomorpholine, pyrrolidone~ piperidine, 2,6-dimethylpiperidine, piperazine and similar saturated monocyclic ni~rogen heterocycles.
R may also represent a point of a~tachment for one or more additional nucleotides at the 3' position. In that case R i8 phosphate, as such additional nucleotides are typically bound through a phosphate group.
As at the 5' position, ~he modified nucleotide ~ 5 may be attached to a support through the 3' position, i.e. through R . When the nucleotide thus attached to a suppoet, R is an anchoring group as will be described below.
R , in the case of deoxyribose, is H; in ~he case of ribose, is OH;, and, during RNA synthesis, is a suitable blocking group which protects the -OH moiety rom modification. Blocking groups useful here generally include those given above for R , and the specific choice of blocking group will be apparent to one skilled in the art. Examples of blocking groups which are preferred at the R position during RNA
synthesis include silyl ethers such as t-butyldimethylsilyl, substituted methyl ethers, o-nitrobenzyl ether, esters such as levulinic ester, and the following pyranyl structures given by Formula 11 ttetrahydropyranyl) and Formula 12 (4-methoxytetrahydropyranyl):
, y l J
~, Formula 11 CH,O ~ O

- Formula 12 -25- 13172~7 A particularly preferred blockiny group is ortho-nitrobenzyl. Additional examples of ~uitable blocking groups may be found in Green, T.W., Pro~ective GrouPs in Organlc Synthesis, New York: Wiley 6 Sons, 19~1.
The modified nucleotide will normally be derivatized with a label in a manner which will allow for detection of complex formation. A wide variety of labels may be used, and one or another label may be selected depending upon the desired sensiti~ity, the equipment available for measuring, the particular protocols employed, ease of synthesis, and the like.
Labels which have found use include enzymes, fluorescers, che~iluminescers, radionuclides, enzyme substrates, cofactors or suicide inhibitors, specific binding pair members, particularly haptens, or the like. The molecule involved with detection may be covalently bound to the modified nucleotide or indirectly bound through the intermediacy of a specific binding pair, i.e. ligand and receptor. Examples of ligands and receptors include biotin-avidin, hapten-antibody, ligand-surface membrane receptor, metal-chelate, etc.
As suggested above, it is preferred that the modified nucleotide be covalently bound to a support at either the R or R positions for oligvnucleotide synthesis. A wide variety of supports may be used, including silica, Porasil*C, polystyrene, controlled pore glass (CPG), kieselquhr, poly(dime~hylacrylamide), poly(acrylmorpholide3, polystyrene grafted onto poly(tetrafluoroethylene), cellulose, Sephade~ LH-20, Fractosil*500, etc.
Depending on the nature of the support~
different functionalities will serve as anchors. A~
(*) Trademark -26_ ~3172~7 noted above, the e ~anchoring" groups are at either the ~ or the 5~ posi~ion, i~e. at either the R or R
position~, respectively. For silicon-containing suppoets, such as silica and glass, substituted alkyl or aryl silyl compounds will be employsd to form a siloxane or siloximine linkage. With organic polymels, ethers, esters, amines, amides, sulfides, ~ulfones and phosphates may find use. For aryl groups, such as polystyrene, halomethylation can be used for functionalization, where the halo group may then be substituted by oxy, thio (which may be oxidized to sulfone), amino, phospho (as ~hosphine, phosphite or phosphate), silyl or the like. With a diatomaceous earth element (e.g., kieselguhr), activation may be effected by a polyacrylic acid derivative and t~e active functionality reacted with amino gcoups to form amine bonds. Folysaccharides may be functionalized with inorganic esters, e.g. phosphate, where the other oxygen serves to link the chain. With polyacrylic acid derivatives, the carboxyl or side chain functionality, e.g., N-hydroxyethyl accylamide, may be used in conventional ways for joining the linking group.
The modified nucleotide of Formula 1, as previously suggested, can be used as a substrate for synthesis of polynucleotide probes. Additional nucleotides may be sequentially added at the 5' position by, for example, the phosphoramidite method of Beaucage and Caruthers, Tetrahedron Lett. 22(20):1859-62 (1981) or the phosphotriester method of Itakura, J. Biol. Chem.
~0 250:4592 ~1975), or the like~ or at the 3' position by Belagaje and Brush, Nuc. A _ ds ~esearch 10 6295 (lg82), or both. The nucleotides which are sequentially added may be unlabeled, or the~ may be modifiad according to Formula 1 and derivatized with a label at the R

. ~27- 13172~7 moiety. Accordingly, one or more labels may be present within a polynucleotide chain rather than at one end.
.This polynucleotide probe includes at least one modified nucleotide having substantially the same 5 structure as that given by Formula 1, i.e. including at least one modified nucleotide having the structure given by Formula 13:

R

(c H2) X
!
NH
N ~ ~ R3 O // ~ N

o R

Foemula 13 wherein R is a reactive gcoup deeivatized with a detectable label, R is an optional linking moieky including an amide, thioether or disulfide linkaqe or a combination thereof, R is selected fro~ the group consisting of hydrogen, methyl~ ~romine, fluorihe and iodine, R is H, OH, or O~ where R is an acid-sensitlve, base-stable protecting group and x is an -z8-1 3 1 ~ 2 07 integer in the range of 1 and 3 inclusive. The polynucleotide probe may have a single label or a plurality of labels, depending ~pon the nature of th0 label and the mechanism for detection. Where the label is fluorescent, for example, a distance of at least 3 to 12 Angstroms should be maintained betwsen fluorescent species to avoid any fluorescence quenching.
Such labeled polynucleotide probes may be used in the assays described in app~icants ' co-pending application Serial No. 807,624, or in any number of other applications, including conjugation with enzymes, antibodies and solid supports. An example of one such use of applicants' novel oligonucleotide probes is in ths detection of a known se~uence of DNA. The probe may be prepared so as to be attached, for example, to a standard latex solid support Ol to an avidin support in the case of biotin-labeled probes. Sample containing single-stranded or double-stranded DNA sequences to be analyzed is caused to contact ~he probe foc a time sufficient for hybridized nucleic acid complexes to form, and any such complexes ace detected by means of the fluorescent, biotin or otherwiss detectable label.
Synthesis of the modified nucleotide: The present invention also relates to a method of synthesizing the novel modified nucleotide of Formula 1. In the preferred embodiment, a pyrimidine nucleotide is provided which has ~he structure of Formula 14 or Formula 15:

1 3 1 72~7 ~ N T oJ\ ~

~4~_~ R40 - ~ / \

o~ IR6 oRS l Formula 14 Formula 15 wherein R is as given above, R and R are hydrogen, and R is OH or H. The 5~ position of the suqar ring -- and the 2' position a~ well if the sugar is ribose rather than deoxyribose -- is then protected against modification during subsequent reaction ste~s by addition of a dimethoxytrityl group (see Example 3) or other suitable protecting group, the additi~n reaction allowed to proceed foe a time sufficient to:ensure substantial completeness. Similarly, the 3' hydroxyl group is protected with a silyl or other suitable functionality (see Examyle 4).
Examples oê particularly suitable protecting groups include those set forth above as "~ ", i.e., substituted methyl ethers, esters, pyranyls and the like.
When the nucleoside is thymine or uracil, or uracil modified at the 5-position by an E
substituent, i.a. a pyri~idine or sub~tituted pyrimidine which has an oxy rather than an amino ~ubstituent at the 4-position, the ~arbonyl is converted to an amine moiety by~ for example. reaction with an activating agent ~uch as l-(mesitylene-Z-sDIfonyl)-tetrazole (MS-~et~ or o~her.

, _30_ 1317207 suitable condensing reagent. Activatiny agents for u~e herein also include other sulfonyl compounds given by the formula El-SO2-Ez wherein E1 i~ tetrazoyl, nitrotriazoyl, triazoyl, imidazoyl, nitroimidazoyl, or s the like, and E2 is an aryl or sub~tituted aryl group such as mesitylene, etc. Another class of suitable activating agents is given by Formula 16:
O O
Il 11 E,-- P - E, Xl P ~X

~--Cl ~ Cl E'ormula 16a ~ormula 16b wherein El is as defined above, and X is a halogen substituent, preferably chlorine. In Formula 16b, El is present in a solution containing the acti~ating agent but is not bound thereto. In general, any activating agent may be used and may include one or more halogen substituents, preferably chlorine, on the ring structure which after reaction can be displaced by ethylene diamine or like reagent. This conver6ion is followed by reaction with an alkyldiamine such as ethylenediamine to give a nucleotide having a -NH-(CH2)XNH2 functionality at the ~-position of the pyrimidine ring (see Examples 5, 6). The free amine qroup so provided is then optionally reacted with caproic acid, an activated caproic acid ester, or with a caproic acid derivative such as 6-aminocaprQic acid, in order to ensure sufficient spacing between the nucleotide and the detectable label to be attached at the R moiety. The ~31- 1 317207 caproic acid or related compound may be labeled prior to attachment (see Example 7) or ~ubsequently.
When the nucleoside i5 cytosine or a S-modified cytosine. i.e. ~ubstituted with an R other than 5 hydrogen, the exocyclic amino functionality can be converted to an N -aminoalkyl or ~ -aminoaryl cytosine by reaction with an aryl sulfo~yl chloride followed by reaction with an alkyl- or aryldiamine (Scheme I). See, e.q., Markiewicz, W.T. and R. Kierzek, 10 7th Intl. Round Table, p~. 32 and 72 (1986).
Alternati~ely, preparation of N4-substituted cytosine may be effected using a bisulfite-catalyzed exchange reaction (Scheme II~. See Schulman, L.H. et al., Nuc.
~cids Res. 9:1203 1217 (1981) and Draper, D.E., Nuc.
15 Acids Res. 12:989-1002 (1984).
Alternatively, where the alkylamine group is more than about 6 carbon atoms long, the free amine group thereof may directly bond to a suitable detectable label.
The synthesis may further include removal of ~he dimethoxytrityl or other protecting groups with acid, followed by, if desired, phosphorylation or phosphitylation of the 3' position in preparation for sequential addition of nucleotides.

"

-31a- 1 31 72rJ7 Schenle I

Nl12 , ~51--R

R--502--Cl ~ R - ~/ ~!

0 \: _~) R
R O

HN (CH2)nNH2 ~ 3 o~5 N fMOCNH(CH2)sCO OH
H2N(cH2~n N~2 ~ Rlo___ , ~ ~' ~

RO

HN(cH2)NHco(cH~)sNHFMoc HN(CH,)NHCOkH,)sl~liFMOC
N~

R O~ DM T-O ~

R O . /N;P'2 ~ ' .
.

~ 31b- 1 31 7207 Scheme I I

~H2 N~2 10 N ~3 11~ N
R R

2 oH2 ~JI 9~ ~ ~ ~ /~

-' ~
-32-. 13172~7 It is to be understood that while the invention has been described in conjunction with the ~referred specific.embodiments thereof, that the foregoing description as well as the examples which follow are intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

-33~ ~3172~7 Exper imenta l Analyte BglII H~V Fragment GATCTCC¦TAGACACCGCCTCAGCTCTGTATCGAGAAG¦CCTITACAGTCTCCTGAG
AGC¦ATCTGTGGCGGAGTCGAGACATAGCTCTTC¦GGAIATCTCAGAGGACTC
~ 1 ~ ~ 2 :

l O CATTGCTCACCTCACC¦ATA¦CTGCACTCAGGCAAGCCATTCTCTGCTGGG¦GGG¦AATTGATG
GTAACGAGTGGAGTGGITATlGACGTGAGTCCGTTCGGTAAGAGACGACCC~CCClTTAACTAC
3 ~ ~
ACTCTAGCTACCTGGGTGGGTA¦ATA¦ATTTGGMGATCCAGCATCTAGGGATCTTG¦TAG¦TA
TGAGATCGATGGACCCACCCAT¦TAT¦TMACCTTCTAGGTCGTAGATCCCTAGAAC¦ATC¦AT
4 ~ + 5 ~ l l 5 AATTATGTTMTACTMCGTGGGTTTM ¦AGAITCAGGCMCTATTGTGGTTTCATATATCT
TTAATACAATTATGATTGCACCCMATTITCTlAGTCCGTTGATMCACCAAAGTATATAGA
6 + + 7 T¦GCC¦TTACTTTTGGAAGAGAGACTGTACTTGAAT¦ATT¦TGGTCTCTTTCGGAGTGTGGATT
A¦CGG¦ MTGAAAACCTTCTCTCTGACATGAACTTA¦TAA¦ACCAGAGAAAGCCTCACACCTAA
+ 8 ~ + 9 2 0 CCCACTC¦CTC¦CAGCCTATAGACCACCAAATGCCCCTATCT¦TAT¦CAACACTTCCGGAAACT
GCGTGAGIGAGlGTCGGATATCTGGTGGTTTACGGGGATAGAlATAlGTTGTGAAGGCCTTTGA
+ ~ 10 ' + 11 ACTGTTGTTAGAC¦GAC¦GGGACCGAGGCAGGTGCCCTAGAAGAAGAA¦CTCCCTCGCCTCGC
TGACAACMTCTG¦CTG¦CCCTGGCTCCGTCCAGGGGATCTTCTTCTT¦GAGGGAGCGGAGCG

TCTGCCTCTAG

~ ~ indicates probed segments ~34- 1317207 I,ab~lling and Capturin~ Probe Sets (Refer to Fig. 1) 3 GACTTCCAAGTTCGTCAACT¦ATCTCTCGCCGAGTCCAGACATACCTCTTC¦ S
A ~ I +
GTGGTGAAAGAGGTTTCTTC¦ATCTCAGAGGACTCGTMCGACTGCACTGG¦
B +
GACTTGCMGTT(`GTCAAGT¦GACGTCAGTCCGTTCGGTMGAGACGACCC¦
A + 3 GTGGTGAAAGAGGTTTCTTC¦TTAACTACTGAGATCGATGGACCCACCCAT¦
B + 4 GACTTGCAAGTTGGTCAAGT¦TAAACCTTCTAGGTCGTAGATCCCTAGAAC¦
A + S
GTGGTGAMGAGGTTTCTTC¦ATTTMTACAATTATGAIIGCACCCAAATT¦
B , 6 GACTTGCMGTTGGTCMGT¦AGTCCGTTGATMCACCAMGTATATAGAA¦
A + 7 CTGGTGAAACACCTTTCTTC¦AATGAMACCTTCTCTCTGACATGAACTTA¦
B ~ 8 +
CACTTGCAAGrTC_CAAGT¦ACCAGAGAAACCCTCACACCTAAGCGTGAG¦
A ~ 9 CTGCTCAAACAGGTTTCTTC¦GTCGGATATCTGGTGCTTTACGGGGATAGA¦
B , 10 GACTTGCAAGTTGGTCMGT¦GTTGTGMGGCCTTTGATGACAACAATCTG¦
A
CTCGTCAAAGAGGTTTCTTC¦CCCTGGCTCCGTCCAGCGGATCTTCTTCTT¦
B , 12 25 ¦ + Labeled Probe + ¦ + Probe Segmants to ItBV
recognition sequences A - Fluorescein label conjugate binding site n - Biotin or DNA conjug~te binding site Label conjugate (A') for DN~ or avidin support:
Fluorescein - S' CTGAACGTTCAACCAGTTCA 3' .
DNA sequence (B'C) bound to solid support:
3' GAAGAAACCTCTTTcACCAcTGTCATCA~AAGGT~rAACCATGTTTCTTGT 5' - ( ) -3S- 1317~07 Biotin conjugate (B') for avidin ~upeort:
Biotin - 5' CACCACTTTCTCCAAAGAAG 3' :..
Preparation of biotin or fluorescein labeled DNA
- 5 (A' or B'):

fH2 1 Fluorescein - NHCNH ~CH2 ) 5CO NE~S
N ~, or 0~ Long chain biotin NHS
( f rom Pierce Chemica l ) 0~ OP03 DNA

N - (2' - aminoethyl) - deoxycytosine - DNA

The analyte is an HBV BqlII fragment as indicated above. (Valenzuela et al. (1981) in Animal Virus Genetics, eds. Fields, B., Jaenisch, R., Fox, C.F., Academic Press, Inc., N.Y., pp 57-70.) A subset of labeling and capturing probes are indicated, where 12 different sequences co~plementary to different sequences present in HBV are provided. Six of the ~BV
complementary sequences are joined to a common sequence (A) for complexing with the lahel conjugate (A'). The other 8iX HBV complementary ~equences are joined to a common sequence (B) for complexing with a biotinylated se~uence (B'~ or a third DNA sequence ~B'C) for-binding to a support. ln Figure l is shown an illustration of .
- :

- `
-36- 13172.07 the final complex involving the H~V 6trand and the various reagents.

Example 1 Labelinq of caProic Acid Derivative (A) Fluorescein --- NHCNH(CH2)5CO~NHS

To 1 mmole of fluorescein isothiocyanate in 5 ml of DMF was added 2 mmole of 6-aminocaeroic acid and 540 ~1 of triethylamine. After 24 h at room temeerature, the ~roduct was isolated by ~reparative thin layec chromatography (Warner and Legg, Inorg. Chem.
15 18:1839 ~1979)). The dried ~roduct was suspended in 10 ml of 1:1 DMF/THF (v/v) to which 1.5 mmole of N-hydroxy succinimide and 1 mmole of dicyclohexylcarbodiimide were added. After 18 h at room temperature the solution was filtered through ~lass wool and diluted to a 0.2M final 20 concentration of ~ with DMF (assuming a 100~ yield from step 1).

Example 2 6-N -(2-Aminoethyl)- DeoxYcvtidine (CH2) 2 NH
(B) N~

HO - ~ O

15 0~
An alkylated derivative of deoxycytidine, N4-(2-aminoethyl) deoxycytidine (B) was prepared f~om pro~erly protec~ed deoxyuridine via tha 4-tetrazoyl derivative as described by Raese and Ubasawa, 20 Tetrahedron Lett. 21:2265 (1984). This latter derivative was converted to B by dis~lacement of the tetrazoyl moiety with ethylene diamine essen~ially a6 described by Sung, J. Or~. Chem. 47:3623 (1982) and Maggio et al., T~trahedron Lett. 25:3195 (1984). The 25 corresponding 5~-DMT-3~-phosphoramidite N4-(2-N-trifluoroacetylaminoethyl) ~eoxycytidine was prepared by blocking the alkylamine with trifluo~oacetic anhydride and then prepa~ing the corresponding N,N-diisopropyl pho6phoramidite as described (Beaucage 30 and Caruther~, supra; McBrida and Caruthers~ Tetrahedron Lett 24:245 (1983)).

.

~ ~ `
-38- 1 ~1 7207 Example 3 Probe PreParation (Fluorescein Labell Synthetic oligonucleotides were prepared by an 5 automated phosphoramidite method a~ describ~d in Warner et al., DNA 3-401 (1984). Purification was carried out according to Sanchez-Pescador and Urdea, DNA 3:339 (1984).
The aminoethyl derivative of deoxycytidine as 10 prepared in Example 2 was incorporated by standard coupling procedures during the oligonucl~otide synthe~is and the purified modified oligonucleotides were used for inco~poration of a fluorescein label as follows. To a dried sample (3-5 OD 260 units) of the aminoethyl 15 deoxycytidine containing oligomer were added 50 ~1 of ~ME and 25 ~1 of the O.M2 stock solution of A
described above. After 18 h at room temperature, the solution was partially purified by Sephadex G-10 chromatography eluted with water, dried and further 20 purified by polyacrylamide gel, as above.

Example 4 Probe PreParation (Biotin Label) Using ~he probes containing aminoethylcytidine as prepared in the previous example, biotin labeling was achieved as follows. The oligonucleotide ~3-5 OD 260 units) was taken up in 50 ~1 O.Ml sodium phosphate, e~
7.0 and 50 ~1 of DMF to which 100-~1 of a DMF
30 solution containing 1 ~g of a "long chain"
N-hydroxysuccinimidyl biotin (Pierce Chemical) was added. After 18 h at room temperature, the biotinylated probe was ~urified a~ described for ~he fluorescein labeled probe.

_39_ i~1 7207 Exam~le 5 Preparation of Solid-SuPported ~NA Probe Fragment B'C (a synthetic 50mer~ was s 5'-phosphoeylated with T4-polynucleotide kinase and ATP
u~ing standard condition~. After gel ~u~ification a~
de&cri~ed above, the oligonucleotide ~as dried by evacuation.
Hydroxylated latex (lOmg; 0.8~: Pandex 10 Labora~ories) was washed wi~h DMSO, then three poL~ions of 40mM MES ~morpholinoethanesulfonic acid), pH 6.0 by cent~ifugation. 1500pmoles of 5'-phosphorylated fragment B'C was taken up in 90ml of 40mM MES and added to the washed support. A solution was prepared to 15 contain 100~g of EDAC in 100ml of MES. After adding 5~1 of the EDAC 601ution and mixing, the reaction mixture was evaporated until 30~1 total remained. The mixture was left at 37C for 18h, then cen~rifuged for 2min at 12,000rpm. The supernatant was discarded. The 20 latex was suspended in 30ml of DMSO, vortexed, 100~1 of water was added, the mixture vortexed for 2min and the supernatant was discarded after centrifuga~ion.
This washing proce6s was cepeated twice. The support was then washed three time5 with 100ml portions of 25 4xSSC, ~2~ then H2O at 37C for 15min (yield 20 picomole~ fragment B'C per mq of latex~.

Example S
As6aY for HBV ~A Usinq DNA Solid Su~port A pBR322 clone containing the entire HBV genome (Valenzuela et al., hnimal Viru8 Genetics, R. Jaenisch, B. Field6 and C.F. Fox, Eds. (Academic Pre6s;: New York) pp. 57-70 (1980)) was cut with BqlII and used as the ~40- 1 3~ 7~.07 analyte nucleic acid. Analyte in lOml of fo~mamide containing 6 picomoles o the labeling and capturing probe sets was heated to 95C for lOmin and cooled to room ~emperature. To thi~ mixture, 60~1 o water, 5 20~1 of 2DxSSC. lOml of 1% NP40 and 2~1 (lO~g) of polyA are added, vo~texed and incubated at 37C for 1~1.
The solid sup~orted DNA probes (8 eicomoles 400vg) is added and incuba~ed for an additional 1.5h.
The mixture i~ centrifuged at 12,000rpm for 2min and ~he 10 supernatant di~carded. The support is washed once by vortexing the pellet into solution wi~h lOOml of 4xSSC, followed by centrifugation. To the washed beads are added a mixture of 4ml of 20xSSC, 2~1 of 1% ~P40, 1~1 (5~g) polyA, 13~1 of water and 6 pico~ole~ of 15 fluorescein labeled probe. After incubation at 37C for 30min, the beads are transfer~ed to a Pandex filter plate, washed four times with lOOml of 4xSSC by vacuum filtration on the 0.2~ cellulose acetate membrane of the plate. The ~ample is vacuumed to dryness and read 20 on the fluorescein channel A ~eXcitation-48s ~emiSSion-525) of the Pandex screen machine.

_ Fluorescence Counts Condition (Averaqe of ~
0.5 pmole HBV 5062 ~/- 345 0.25 pmole HBV 4117 ~f- 262 No Analyte 3197 ~f- 520 No Biotinylated Probe 3856 +~- 642 Example 7 Assay for H~V DNA Usinq ~vidin SupPort Experiment 7a:
Analyte was mixed and incubated with the labeling and capturing probes as above. Biotin labeled probe (12 picomoles~ in 5~1 a20 was th~n added, vortexed and incubated at 37C for 30min. To the mixtu~e, 20ml of a 0.25% (w/v) 0.8~ avidin latex 10 (Pandex Laboratories) in lxPBS is added and incubated at 37C for lh. The mixture is washed~ incubated with fluo~escein probe, washed and read on ~he Pandex screen machine as described above.

Fluorescence Counts Condition (Averaqe Of 4 ?

0.5 picomole HBV 4052 ~ 62 0.25 picomole HBV2644 +/- 397 0.10 ~icomole HBV1956 +~- 173 No Analyte 1641 ~/- 370 No Biotinylated Probe1631 +/- 474 .. .. . _ . . . .. _ _ . _ . _ . _ _ _ 30 Experiment 7b:
The HBV plasmid ~as sonicated to an averaya size of 500b~. The denaturation and hybridization were carried out as above exce2t that 30 picomoles of label;ng and capturing probes were used and a 5h ~z-annealing was employed. After incubation with 30 2icomoles of biotinylated probe (2h}, 50~1 of 0.25 avidin bead~ were added and incubated ~1.5h).
fluorescein erobe was added and incubation was carried 5 out for lh followed by washing and reading on the Screen ~achine as described above.

TABL~ 3 Fluorescence Counts Condit;on (~veraae of 4 0.5 picomole HBV 5748 +/- 244 0.4 eicomole HBV 5352 +/- 331 0.3 picomole HBV 4716 +/- 243 0.2 picomole HBV 4071 ~- 243 0.1 picomole HBV 3320 +/- 27t No Analyte 1679 ~/- 167 No Biotinylated Probe 1716 +/- 177 It is evident from the above results that a highly specific ~ensitive assay for s~ecific nucleic acid ~equences is provided. ~eagents can be readily prepared to the sequence of interest and with a few 30 si~ple manipulative ste~, the presence or ab~ence o~ a sequence in a 6ample determined. The ~ethod is versatile in per~itting a wide variety o~ labels which can be readily determined by conven~ional equipment.
Peobes can be synthe~ized to the de~ired leng~h and - ~~3 l 3~ 7~07 easily linked to the label oe a ~up~oet. Universal sequences can be prepared for the label and binding to the su~pqct. Various protocols may be employed where more or les6 rigorou6 ~emoval of background interference 5 is a~hie~ed depending upon the requiremen~s of the assay.

ExamPle 8 5'-DimethoxvtritYl-2'-Deoxyuridine 1 s Ho`,,NI,~
(C~ I
DMTr O ~ o ~J
y OH

To 2-Deoxyuridine (10 g, ~4 mmole) dried by coevaporation of pyridine and suspended in pyridine (lOO
ml) was added 18.4 g (54 mmole) 4,4'-dimethoxytrityl chloride (DMT Cl). The reaction was allowed to proceed foc 18 h at room temperature, and 100 ml methanol was 30 added to deactivate excess DMT-Cl. Most of the pyridine was then eemoved in vacuo, and the re~idue~ dis601ved in 500 ml ethyl acetate, was washed with saturated aqueous NaHC03 (3x5~0 ml). The organic phase was deied ove~
- solid Na2S04 and evaporated to d~yness. The ~esidue . ~

-~- 13172(:~7 was purified by fla~h chromatography on ~ilica gel ~o give 18.0 g (77%) of 5I dimethoxytrityl-2'-deoxyuridine (C) ., Example g 5~-0-(4 4'-Dimethoxytrityl)-3'-t-Butyldimethyl~ilYl-2'-DeoxYuridine , ~
J~
~N 1¦
o 5 15 (D)DMTrO I ~

Si-(t-Bu)Me 2 To 18 g (34 mmole) of C in 200 ml DMF was added 25 imidazole ~5.8 g, 85 mmole) with rapid stircing to as~u7e complete di~solution. t-Butyldimethylsilyl chloride ~7.65 g, 51 mmole) dissolved in a small volume of ~MY wa~ added drop~7ise with stirring and the reac~ion was allowed to proceed in the dark ~or 18 h at room 30 temperature. The reaction mixture was diluted with ethyl acetate ~250 ml) and extracted with NaHCO3 (3x250 ~1~. The organic phase was dried oYer Na2S04 and evaporated to drynes~. The residue was purified by flash chroma~ography on silica gel to give 15.0 g (68%

' .

-45_ 1 317207 yield) of 5'-0-(4,4'-dimethoxytcityl-~'-t-butyldimethyl-silyl-2'-deoxyuridine (D).

Example 10 4-(1,2,3,4-Tetrazol_l-yl~=~5'-(4,4'-DimethoxYtrityl)-3'-t-ButyldimethYl-silvl-~-D-2'-DeoxvribosYll Pyrimidine-2~1H)-one ~N-\N~
N
(E) 0 ~ N

DMTrO -Si-(t- E~u)Me2 To 15.0 g (23 mmole~ of D, dried by coevaporation of pyrimidine and dissolved in pyrimidlne (50 ml) was added diphenylphosphate (2.9 g, 11.5 mmole) dissolved in pyrimidine ~5 ml). 1-(Mesitylene-2-sulfonyl)-tetrazole (MS-~et) (15.5g, 61.5 mmole) ; dissolved in pyrimidine (45 ml) was added and the reaction 30 mixture allowed to proceed in the dark for 18 h at room temperature. To the dark brown reactio~ mixture was - - added 25 ml water. Aftar 30 min. the produc~ was concentrated under reduced pressure. The residue was . dissolved in 250 ml methylene chloride, washed with an aqueous Na~lC03 solution (3x250 ml), dried over ~a2S04, and the ~olven~ wa~ removed under reduced pressure in the p~e~ence of toluene. The ~e6idue wa~
pueified by flash chromatography on silica gel to give 10.0 g (62%) of 4-(1,2,3,4-Tetraæo.l-l-yl~-[5'-(4,4l-dimethoxytrityl)-3'-t-butyldimethyl~ilyl-~-D-2'-deoxy-ribosyl]- pyrimidine-2(1H)-one (E) ExamPle 11 4-N-(2-Aminoethvl)-5'-DimethoxYtritYl-3'-t-Butyld,imethylsilyl-2'-DeoxYcytidine -I -(l H2)2 NH

DMTr O ~

05i-~-BU)Me2 To a solution of ethylene diamine (9.3 ml, 143 mmole) in dioxane (100 ml) cooled .to 5C was added E
~0 (10.0 g, 14.3 mmole) and left or one hou~. The ~olvent wa~ removed a~ reduced pressure and the re~idue was coevaporated with toluene to ~emove exces6 ethylene diamine. -The product wa~ purifiea by chromatography on a silica gel column, elu~ed with 1~-20~ methanol in methylene chloride to give 7.15 g (7S%) of 4-N-(2-aminoethyl)-5'-dimethoxytrityl-3'-t-butyldimethyl-6ilyl-2'-deoxycy~idine (F). The product was ~hown ~o react po6itively with ninhyd~in, confirming the presence 5 of a free amine moiety.

ExamPle 12 N -(~-FMOC-6-AminocaproY)-2-Aminoethyl~-5'-Dimethyltrity1-3'-t-Butyldimethylsilyl-2~-Deoxyc~tidine o~ (~H2)s-NH-FMoc NH

(CH2)2 NH
(G) N~3 O'~N
DMTrO-- ~

oSi(t-Bu) Me2 PMoc = ¢~ 3 ' -48- 1 31 72~7 s To a ~olution of F (6.5 g, 9.6 mmole) in pyridine (50 ml) was added N-FMOC-6-aminocaproic acid (4.26 g, 12 mmole) (FMOC rep~esented by structure H~ and DCC (2.96 g, 14.4 mmole). After 3 h, the reac~ion was 10 complete as judged by tlc (silica in 10~ ~e~hanol/
methylene chloride). Pyridine was removed at ~educed pressure. The residue was extracted with ethyl acetate, insoluble dicyclohexylurea (~CHU) ~iltered off and the solvent removed. l'he product was isolated by silica gel 15 chromatography eluted with 4~ methanol in methylene chlo~ide affording 7.3 g (70%) of N -(N-FMOC-6-amino-caproyl-2-amino-ethyl~-5'-dimethyltrityl-3'-t-butyldi-methylsilyl-2'-deoxycytidine (&~.

Example 13 o~
~C -(CH~)sNH ~ FMOC
NH
I

(CH2)2 NH
(I) N~

O ~ N
3 DMT~O ~O ~) r OH

49 13172~)7 A ~olution of tetrabutylammonium ~luo~ide (15 mmole, 15 ml of a lM solution in THF) and aqueou6 HF
(1.05 ml of a 50% aqueous 601ution) were mixed and dried by coevapo~ation of pyridine. The residue was dissolved 5 in eyridine (15 ml) and added to G (7.2 g, 7.3 mmole~
which wa6 disfiolved by sonication. ~fter 18 hours at 4~C the ~e~ction mixture was diluted with 200 ml methylene chloride. Concentrated aqueou~ NaHC03 was ca~efully added followed by solid NaHCO3, added 10 gradually ~o a~ to neutralize the HF/pyridin~. Af~er drying over Na2SO4, the o~ganic phase was concentrated to an oil, which was subjected to silica gel chromatography. The product N -(N-FMOC-6-amino caproyl-2-aminoethyl)-5l-dimethoxytrityl-' deoxycytidine (I) wa~ elu~ed with 5-6% methanol in methylene chloride to give an 86% yield (6.0 g).

Example 14 C-(CH?)sNH--FMoc NH
(cH2) I

(J) NH
N~
o~lN
DMT~O--~0~ CH, \lJ ~OCH, CH--CH
O- p N ~ ~CH3 CH

- ` ' " c t"

-so 1317207 To 5.1 g (5.7 mmole) of I in methylene chloride containing (diisopropylethylamine) was added CH, - - S C / C H--C
( K ) 'C~
CH, 10 (chloro-N,N-diisopropylaminomethoxy phosphine, 1.3 ml tl.2 eq.], K) at 0C under argon. After 1 hr, ethyl acetate (200 ~1) wa~ added and washed with BO% saturated aqueous ~odium chloride; afte~ drying of the organic ~hase over ~a2S04, the product in methylene chloride 15 was added dropwise to hexane at -40C ~o precipitate 4.43 g ~75~) of J.

Example 15 Sy~thesis of Horseradish 20Peroxida6e lHRP~: DNA Conjuqates Sequence 1 (5'-~LCA]CTGAACGTTCA~CCAGTTCA-3') whera LCA = N4(Ç-aminocaproyl-2-aminoethyl)-deoxy cytidine) wa~ synthesized chemically and purified as 25 described el6ewhere (warner, et al. (1984) DNA 3, 401).
To 10 OD 260 units dissolved in 50 ~1 of water weee added 10 ~1 of 1.0 ~ sodium borate, pH 9.3, and 500 ~1 of difitilled dimethylformamide containing 20 mg of p-phenylene diisothiocyanate. The solution was vortexed 30 and 8et fOL 2 hr at room temperature in the dark.
Approximately 3 ~1 of n-butanol was then added. After vortexing, adding 3 ml of water. and vortexing again, the tube wa~ centrifuged and the yellowi6h upper layer -disc~rded. The extraction proce~s was repeated with ~iubsequent n-butanol addi~ions until an final volume of approximately S0 ~1 was obtained. The butanol was removed by evacuation, then 10 mg of HRP in 200 ~1 of o.l M borate, pH 9.3, was added. The mixture was - 5 vortexed, ~hen ~e~ at room tempera~ure overnight in the dark.
Separation of the HRP-DNA conjugate from ~ree enzyme and DNA was achieved on a 7~ polyacrylamide gel.
The 250 ~1 reaction mixture was quenched with 100 ~1 10 of 25% glycerol, 0.5% SDS, 0.5% bromophenvl blue, 2.5 mM
EDTA. The solution was then distributed into 10 lane~
of a 20 x 20 0.15 cm gel and run at 60 mAmps under standard conditions (Maxam, A., and Gilbert, W., (~980) Methods in Enzymol fi5, 499-560) until the bromophenol 15 blue was about 2/3 down the gel. The gels were set on Baker*F-254 silica 60 plates that ~ad been covered with Saran Wra~ (Dow) and examined with a handheld W -short wavelength lamp held above. Pictures of the W -shadowed bands were taken with a Polaroi~ MP-4 camera system - 20 fitted with a Kodak*No. 59 green filter. after which the bands were cut out with a razor blade. The bands we~e put into a 10-ml Bio-Rad~polypropylene econo~columns to which 3 ml of 0.1 M sodium phosphate, pH 7.5, was added, then set at room tempera~ure overnight.
The conten~s of the column were filtered through the ~rit at the column bottom into an Amicon Centricon*microconcentrator that had been washed twice with distilled water. The HRP-~NA conjugate was then concentrated by centrifugation at-35~0 rpm and washed 30 twice with 1~ PBS al~o by centrifugation. The final solution was then stored at ~C.
- (*) Trademark -52- l 31 7207 ExamPle 16 AssaY for HBV DNA Usinq HRP~DNA Probe and a Biotin lated Probe Bound ~o an_Avidin Bead _ 5 ~iotin labeled probe (B'; lOOO pmoles in 66.7 ~l of water) was combined with 5 ml of a 0.25~ (w/v) solution of 0.8 ~ avidin beads (Pandex laboratories), 1 ml of 20x SSC, 0.5 ml of l~ NP~O and 0.6 ml of l mg/ml polyA. After l h at 37C, the beads were washed twice lO by cent~ifugatio~ with 4x SSC, 0.1% NP40 then s~o~ea in 2.5 ml of this solution. The HBV analyte (described above) in 3 ~l water was diluted into lO ~l of 4x SSC, 1% SDS, 0.5 M NaOH and 1.5 pmoles of the labeling and capturing p~obe ~ets. The mixture was heated to 15 95C for lO min., cooled on ice and neutcalized with 5 ~l of l M acetic acid, then lO ~l of the biotin probe beads we~e added and the solution wa6 incu~at~d at 37C for l h.
The beads were washed twice by centrifuga~ion 20 with 4X SSC, 0.1% NP40, then taken up in 50 ~l of 0.1%
NP40, l mg/ml poly~, lO mg/ml BSA, lX PBS con~aining l pmole of HRP-DNA conjugate and set a 37C for ~ h. The beads were washed with 0.1% NP40, lX PBS three times then transferred in 50 ~l to a microtiter dish. To 2~ each well, 50 ~l of fresh OPD solution (~8 mg OPD
(O-phenyle~ediamine), 20 ~l f 30% H202 in 10 ml of 50 mM sodium citrate pH 5.0) was added, mixed and set 5 min. a~ 37C. The absorhances wece recorded on a microtiter plate reader. Contcol hybridizations 30 contained no HBV analyte.
.

_53_ 1 31 7207 Table 4 Condition Ab~orbance Readinq pmole >2 -~ 5 0.1 pmole . >2 - 0 . 01 pmole 0 . 88 i 0 . 23 fmole 0 . 20 i 0 . 05 0.1 fmole 0.07 i 0.03 N0 P.N~LYTE 0 . 01 * O . 01 :

.

Claims

Claims 1. An assay method for detecting a nucleic acid sequence in a sample, employing two sets of reagents: a first labeling set; and a second capturing set, said method comprising:
combining in the liquid medium under binding conditions for complementary pairs, said sample containing analyte in single-stranded form, members of said labeling set of reagents comprising:
(a) one or a plurality of labeling nucleic acid probes, different probes having a different first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-label conjugate, wherein said label provides, directly or indirectly, a detectable signal; and members of said second capturing set of reagents comprising:
(c) one or a plurality of capturing nucleic acid probes, different probes having a different second analyte complementary sequence and a second capturing reagent recognition sequence; and (d) a nucleic acid sequence complementary to said second recognition sequence-a first member of a specific binding pair conjugate; and separation means;
with the proviso that, said nucleic acid sequence complementary to said second recognition sequence may be bound to said separation means eliminating said specific binding pair, so that (d) is not employed;

separating label into a bound phase and an unbound phase by means of said separation means; and detecting the amount of bound or unbound label as determinative of the presence of said analyte.

2. A method according to Claim 1, wherein said nucleic acid sequence complementary to said second recognition sequence is bound directly to said separation means, and said analyte, (a), (b), and (c) are combined together for a time sufficient for nucleic acid complexes to form, followed by the addition of said nucleic acid sequence complementary to said second recognition sequence bound to said separation means.

3. An assay method for detecting a nucleic acid sequence in a sample, employing two sets of reagents: a first labeling set; and a second capturing set, said method comprising:
combining in the liquid medium under binding conditions for complementary pairs, said sample containing analyte in single-stranded form, members of said labeling set of reagents comprising:
(a) one or a plurality of labeling nucleic acid probes, different probes having a different first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-label conjugate, wherein said label provides, directly or indirectly, a detectable signal; and members of said second capturing set of reagents comprising:
(c) one or a plurality of capturing nucleic acid probes, different probes having a different second analyte complementary sequence and a second capturing reagent recognition sequence; and (d) a nucleic acid sequence complementary to said second recognition sequence-a first member of a specific binding pair conjugate, for a time sufficient for nucleic acid complexes containing analyte, label and said specific binding member to form;
combining any of said complexes with a separation means conjugated to a second complementary member of said specific binding pair under conditions resulting in specific binding pair under conditions of complex formation and separation of label into a bound phase and an unbound phase; and detecting the amount of bound or unbound label as determinative of the presence of said analyte.

4. An assay method for detecting a nucleic acid sequence in a sample, employing two sets of reagents: a first labeling set; and a second capturing set, said method comprising:
combining in a liquid medium under annealing conditions for complementary nucleic acid sequences, said sample containing analyte in single-stranded form, members of said labeling set of reagents comprising:
(a) a set of labeling nucleic acid probes, each subset having a different first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-fluorescer or enzyme label conjugate; and members of said second capturing set of reagents comprising:

(c) a set of capturing nucleic acid probes, each subset having a different second analyte complementary sequence and a second capturing reagent recognition sequence; and (d) a nucleic acid sequence complementary to said second recognition sequence-hapten conjugate, for a time sufficient for nucleic acid complexes containing said analyte, label and hapten to form;
combining any of said complexes to a particle conjugated to a receptor for said hapten under conditions resulting in hapten-receptor complex formation and separation of label into a bound phase and an unbound phase; and detecting the amount of bound or unbound label as determinative of the presence of said analyte.

5. A kit for detecting a nucleic acid analyte comprising:
(1) members of a labeling set of reagents comprising:
(a) a labeling nucleic acid probe having a first analyte complementary sequence and a first label reagent recognition sequence; and (b) a nucleic acid sequence complementary to said first recognition sequence-label conjugate, wherein said label provides, directly or indirectly, a detectable signal; and (2) members of a second capturing set of reagents comprising:
(c) a capturing nucleic acid probe having a second analyte complementary sequence and a second capturing reagent recognition sequence;

(d) a nucleic acid sequence complementary to said second recognition sequence-a first member of a specific binding pair conjugate; and (e) support means conjugated to a second complementary member of said specific binding pair.
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