JP2002518060A - Nucleotide detection method - Google Patents

Abstract

  (57) [Summary] Method for identifying selected nucleotides in a first nucleic acid using a mobile solid support, and improving the use of mobile solid support-based readout techniques to detect nucleic acid polymorphisms in a target nucleic acid A new reading method is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention provides a rapid method for detecting single nucleotide polymorphisms (SNPs) in a nucleic acid sample. The present invention further provides novel readout methods that improve the use of mobile solid support-based readout techniques to detect nucleic acid polymorphisms in target nucleic acids.
The method is useful for genomic DNA as well as for SNPs in amplified DNA, RNA, etc.
A variety of objectives, including genotyping (eg, for detection of disease mutations and pedigree determination in humans and other animals), detection and identification of pathogens, and differential gene expression because they can be used to detect Will be useful. The invention further provides nucleic acid identification methods using run-off sequencing reactions of relatively short portions of nucleic acids. The method can be used, for example, to identify ESTs from a small portion of the ESTs and to analyze nucleotide polymorphisms. This reaction can be multiplexed to increase the data reading capacity.

[0002]

BACKGROUND OF THE INVENTION

Methods for detecting single nucleotide polymorphisms typically include a hybridization reaction. For example, a method of performing SNP analysis based on Luminex FlowMetrix involves differential hybridization between a PCR product and two different FACS analysis beads. Currently, FlowMetrix systems are uniformly sized 5 micron polystyrene stained with eight concentrations of two dyes (orange and red).
Consists of divinylbenzene beads. The matrix of the two dyes at eight concentrations is
The FACScalibur, suitably modified using a Luminex PC computer board, will give beads (8 ² ) of 64 different colors, each identifiable. Luminex
In SNP analysis, short (approximately 18-20 bases) "target" oligodeoxynucleotides (oligos) are covalently attached to the beads. The nucleotide located at the center of the target oligo encodes a polymorphic base. A pair of beads is synthesized, with each bead of the pair being bound to one of the polymorphic oligonucleotides. PCR of the DNA region around the SNP to be analyzed
Create a CR product. Establish conditions for selectively hybridizing the PCR product with beads encoding the exact complement. In one format ("without competitors"), the PCR product itself incorporates a fluorescein dye and FACScali
The increment of fluorescein staining on the beads measured during the bur run indicates hybridization. In another format ("with competitor"), the beads hybridize with the competitor to the PCR product. In this case, the competitor itself is labeled with fluorescein. And it shows that the deficiency of fluorescein replaced by the unlabeled PCR product successfully hybridized. In SNP analysis,
The case with competitors "has been identified as being better identified.

[0003] Labeled Genetic Bi, a method for classifying single nucleotide polymorphisms in DNA
Genetic Bit Analysis: Solid-phase method for classifying single nucleotide polymorphisms Nikiforov TT; Rendle RB; Goelet P; Rogers
YH; Kotewicz ML; Anderson S; Trainor GL; Knapp M R. NUCLEIC ACIDS RE
SEARCH, (1994) 22 (20) 4167-75]. In this method, first, a specific DNA fragment containing a polymorphic site is amplified by polymerase chain reaction (PCR) using a regular DNA fragment and one phosphorothioate-modified primer. Double-stranded PCR product
Single-stranded by treatment with 7 genes and 6 exonuclease enzymes and hybridized with immobilized oligonucleotide primers to capture individual wells of a 96-well polystyrene plate. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent to the polymorphic site of interest. E. coli
li) Klenow fragment of DNA polymerase I or modified T7 DNA polymerase (S
Using equenase), the 3 'end of the capture oligonucleotide is extended by one base utilizing a mixture of biotin-labeled, fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Next, using an antibody conjugate of alkaline phosphatase and horseradish peroxidase, the nature of the extended base by ELISA is examined. This dissertation also
The biochemical characteristics of this method are described in detail. This semi-automated method, called Genetic Bit Analysis (GBA), has been used extensively for thoroughbred proof of Thoroughbred using 26 predetermined sets of biallelic polymorphisms in the horse genome. . Furthermore, mini-sequencing using immobilized primers has been used to detect mutations in PCR products [Minisequencing: A Specifi
c Tool for DNA Analysis and Diagnostics on Oligonucleotide Arrays. Pasti
nen, T. et al. Genome Research 7: 606-614 (1997)].

[0004] The effect of phosphorothioate linkages on the hydrolytic activity of the 5 '→ 3' double stranded specific T7 gene 6 exonuclease to improve GBA has been studied [phosphorothioate for the preparation of single stranded PCR products] Use of primers and endonuclease hydrolysis and their detection by solid phase hybridization Nikiforov TT; Rendle RB: Kotewicz ML; Rogers Y H. PCR METHODS AND
APPLICATIONS, (1994) 3 (5) 285-91]. One phosphorothioate residue at the 5 'end
The contained double-stranded DNA substrate has been found to be hydrolyzed by this enzyme with the same efficiency as the unmodified form. However, this enzyme activity was completely inhibited by the presence of the four phosphorothioates. Based on these results, a method for converting a double-stranded PCR product into a full-length single-stranded DNA fragment has been developed. In this method, the PC
One of the R primers contains four phosphorothioates at the 5 'end and the opposite primer strand is unmodified. After amplification, the double-stranded product is treated with T7 gene 6 exonuclease. The phosphorothioated chain is protected from the action of this enzyme, while the opposite chain is hydrolyzed. When the phosphorothioated PCR primer is 5 ′ biotinylated, a single-stranded PCR product can be easily colorimetrically detected after hybridization with an oligonucleotide probe immobilized on a microtiter plate. A simple and effective method of immobilizing relatively short oligonucleotides to microtiter plates having a hydrophilic surface in the presence of salts has also been described.

DNA analysis based on template hybridization (or hybridization and enzymatic treatment) with a surface-bound oligonucleotide array is well suited for high-density, parallel, low-cost, and automatable processing. Fluorescence detection applied to non-electrophoretic DNA diagnostics Ives. Jeffrey T .; Roger
s, Yu Hui; Bogdanov, Valery L .; Huang, Eric Z .; Boyce-Jacino, Michael; G
oelet, Philip LLC, Proc. SPIE-Int. Soc. Opt. Eng., 2680 (Ultrasensiti
ve Biochemical Diagnostics), 258-269 (1996)]. Direct fluorescence detection of labeled DNA has the advantages of linearity, wide dynamic range, detection of multiple analytes, ease of processing, and safe handling at a reasonable cost. Molecular Tool has filed a patented enzymatic method of solid-phase genotyping for DNA processing in 96-well plates and microscope slides. Detection of fluorolabeled GBA dideoxynucleotides requires a detection limit of about 100 mol / μm ² . A commercially available plate reader detects about 1000 mol / μm ² , and if an experimental setting is performed using an argon laser or a thermoelectrically cooled CCD, a signal that is about one order smaller can be detected. Current limitations are due to glass fluorescence. Dideoxynucleotides labeled with fluorescein, eosin, tetramethylrhodamine, Lissamine and Texas Red have been identified and have been investigated for photobleaching, quenching and indirect detection with fluorescent substrates.

Although SNP analysis by hybridization is an effective method, it has several disadvantages. These disadvantages include: i) the need to synthesize two targets and possibly two competing oligonucleotides for each allele pair; ii) hybridization parameters (buffers) that are effectively distinguished between the alleles. Content, temperature, time) and iii) avoidance of regions containing secondary structures that may affect their hybridization parameters.

The current limitations of the GBA method described are: i) limited density achievable with a two-dimensional solid phase surface; ii) photobleaching; iii) autofluorescence of glass and plastic supports; iv). Difficulties in tightly binding oligonucleotides to glass, and v) the cost, ease and flexibility of creating new immobilized arrays.

The present invention overcomes the drawbacks described by GBA single base chain extension (SBCE), which exploits the potential matrix-like performance of mobile solid support systems with multiple dye / concentration capabilities (eg, FlowMetrix system). A new system for using is provided. The present invention further provides methods for improving the detection of reaction products obtained from such polymorphism identification methods. Various detection methods described herein and known in the art can be enhanced by using the present detection methods. Such a method provides a means of using any of the predetermined beads used with each of the many primers, and can provide a time- and cost-effective read format in conjunction with the present invention. This readout method also covers SBCE, OLA, and cleavage reaction / signal emission (Invader met
hods, Third Wave Technologies).

[0009]

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to dyes, radiolabels, magnetic tags or Quantum Dot® (Quan
Mobile solid supports, such as beads with detectable tags such as Tum Dot, can be used for SBCE, OLA or cleavage reaction / signal release (Invader methods, Third Wa
ve Technologies, Madison, Wisconsin) for direct reading on nucleic acids attached to a mobile solid support, or indirect reading (liquid phase) which is then supplemented by an intermediate nucleic acid such as by a zip code attached to a mobile solid support. Provided is a method for use in any nucleic acid reading method, wherein the read product is then passed through its mobile solid support over a detector (such as a laser detector) or the detector is mounted on a mobile solid support. Analyzed on the selected platform, such as by passing over a support.

The present invention provides a novel SNP reading system using a coded mobile solid support that takes advantage of the potential matrix forming capabilities of the mobile solid support. In one embodiment, the system uses GBA single base chain extension (SBCE). In another aspect, the system utilizes an oligonucleotide ligation assay. In yet another aspect, the system uses an enzyme or chemical to modify or endonuclease the mismatched base at the polymorphic site to delete the binding reporter, or the modification is for identification of the modification. An enzymatic or chemical readout method is used as a labeling means. Thus, in a further aspect, the system comprises Invader methods, Third
d Wave Technologies) (for example, Marshall et al. J. Clin. Microbiol. 35 (12)
: 3156-3162 (1997); Brow, et al. J. Clin. Microbiol. 34 (12): 3129-3137 (1
997)). In another embodiment, the reading is fluorescence energy transfer (FE).
T) is used in combination with the disappearance of fluorescence.

[0011] In the cleavage enzyme readout, target nucleic acids (eg, PCR products or genomic DNA
) Hybridizes with both the complementary invader probe and the signal probe, and the cleavage enzyme recognizes the specific structure formed between the target nucleic acid and the invader probe and the signal probe, and cleaves the signal probe at the branch point Thereby emitting a detection signal. Then another signal probe can bind to this nucleic acid and the cleavage reaction starts anew. This process is repeated many times, thereby increasing signal amplification. The essence of working cleavage lies in the presence of overlapping bases of the signal base with the invader probe. An improved version called Invader Squared performs two invaders simultaneously. For the first invader reaction, S
Involving the use of NP-specific target DNA, the resulting cleavage product becomes functional in a second invader reaction of the universal signal probe with the universal complementary target DNA. After the second Invader assay, a linear signal amplification of over 10 ⁶ signals / target / hour is obtained.

The present invention further provides a first complementary region complementary to a target nucleic acid and a first complementary region complementary to a capture oligonucleotide linked to a mobile solid support.
A novel oligonucleotide for improving the use of mobile solid support-based readout techniques to detect nucleic acid polymorphisms in target nucleic acids using a target oligonucleotide having a 'second complementary region Provides a reading method. The improved method is applicable to any of a number of methods for identifying nucleic acid polymorphisms, such as the oligonucleotide ligation assay (OLA) or single base chain extension (SBCE) described herein. The method can be used to detect SNPs in genomic DNA as well as in amplified DNA, RNA, etc., and can therefore be used for genotyping (eg, for detecting disease mutations or determining the pedigree of humans and other animals), It will be useful for a variety of purposes, including detection and identification, and differential gene expression.

The present invention further provides for the development of a simple method for multiplexing short sequencing reads (about 16 bases) in the same lane. One application to which this method can be applied is
There is a high throughput yeast two-hybrid analysis. In this analysis, it is desirable to use a large database to sequence short regions of interacting proteins and then look for hits. Since each analyzed decoy produces about 100 hits, this method was required to increase the analysis efficiency and was therefore developed.

The present invention relates to amplified DNAs such as genomic DNAs and PCR products, cDNAs, c
It can be used to analyze RNA, nucleic acid samples containing restriction fragments, or any other nucleic acid sample desired. One of the methods described herein uses genomic DNA
Typically, genomic DNA is treated to reduce the viscosity of the DNA so that primers or probes are in good contact with the target region of the genomic DNA. Such reduction in viscosity can be achieved by any desired method known to those skilled in the art, such as DNase treatment or, preferably, light shearing of the genomic DNA. Amplified DNA may be obtained using any of several known methods. There are many sources of genomic DNA, including any tissue, organ or cell selected, depending on the purpose for performing the method. Oligonucleotides can be made, for example, by amplification or de novo synthesis. Complementary nucleic acid, ie cRNA
(The DNA is primed by T7 RNA polymerase / specific sequence primer fusion, and then T7 RNA polymerase is added to amplify the first strand to generate cRN
A) and the cDNA can be obtained by standard methods known in the art.

Accordingly, in the present method, “nucleic acid” includes, for example, oligonucleotide, 16s ribosomal RNA, PCR product, DNA fragment, RNA molecule, cDNA molecule or cRN.
A molecule containing any of the A molecules, the nucleic acid primer is an oligonucleotide, a PCR product,
DNA fragment, RNA molecule, cDNA molecule or cRNA molecule. As an example, the primer or probe is often an oligonucleotide, but the source of the primer or probe is not particularly limited here. As used in the claims, "a" and "an" may be more than one, depending on the context in which it is used.

In the basic SBCE method, a single oligonucleotide is attached to a detectably tagged mobile solid support, such as a bead or rod, which is supported by a FACS. It is preferable to use a system that can perform processing for tag detection immediately after a desired reaction has occurred, such as a system of a type. For example, if the support is finally fixed at a predetermined position prior to detection, “Tentagel” (“Octopus”
) Can be used and then fixed in place prior to detection. Any desired tag can be used, such as a fluorescent tag, a radioactive label, or a magnetic tag. Other detection systems can be used, but preferably the mobile solid support is passed over a detection device such as a laser detection device that can detect and identify the selected tag or label (see, eg, PCT Publication WO 9714028). Further, a detection system in which the movable solid support is immobilized on a two-dimensional surface after any of the reactions, and a detection device such as a laser detection device is passed over the immobilized movable solid support can also be used. The mobile solid support may be made of any useful material, such as polystyrene-divinylbenzene. The detection of the mobile solid support and any nucleic acids or nucleotides bound thereto is performed by Lu
It can be performed by a method based on FACS such as the minex FlowMetrix (trademark) system.

[0017] In a typical assay, the oligonucleotide is designed such that the 5 'end is attached to the bead. Depending on the assay being performed, the 3 'base ends with the nucleotide selected for the polymorphic base. For example, 3 'of this primer or probe
The base may end with a nucleotide 5 'to the polymorphic base or end with a base corresponding to the polymorphic base. Oligo length is not important in the SBCE method.
It must be long enough to aid hybridization with nucleic acid samples such as PCR products generated from surrounding areas. Depending on the assay performed,
Primers or probes can be designed to require a correct match, or they can be designed to tolerate some mismatches upon initial hybridization with a sample nucleic acid.

In a typical assay, nucleotides capable of chain termination are used. Such chain termination is a chain termination event that occurs before the occurrence of the same labeled base in the target sequence occurs again. Such nucleotides are known in the art and include, for example, dideoxynucleotides (if a polymerase is used in the extension reaction), thiol derivatives (if a polymerase is used in the extension reaction), 3 'deoxynucleotides (when a reverse transcriptase is used in the extension reaction). 3 'deoxyribonucleotides (if reverse transcriptase is used in the extension reaction). Any of these nucleotides can be, for example, dinucleotides, trinucleotides or longer nucleic acids. Thus, for example, one may have a bank of dinucleotides or longer nucleic acids, such as having any nucleotide at more than one position within the bank.

Therefore, in the present invention, it is typical that the labeling step is performed in a liquid phase (hence, efficient hybridization is obtained), and the analyzing step is performed in either a liquid phase or an immobile solid support. Can be.

Accordingly, the present invention is a method for identifying a selected nucleotide in a first nucleic acid, comprising: (a) attaching the first nucleic acid to a detectably tagged mobile solid support; 5
A nucleic acid primer, which is linked at the terminus and contains a region complementary to the section of the first nucleic acid immediately 3 ′ to and adjacent to the selected nucleotide, to the first nucleic acid and the nucleic acid primer. (B) performing a primer extension reaction under primer extension conditions using the hybridization product and a detectably labeled end-of-chain nucleotide. And (c) detecting the presence or absence of a label incorporated in the hybridization product, wherein the presence of the label indicates that the labeled nucleotide is incorporated in the hybridization product, and The characteristic indicates a characteristic of a nucleotide that is complementary to the selected nucleotide; Thus identifying a selected nucleotide in the first nucleic acid.

[0021] In certain embodiments, the primer is designed such that its 3 'base ends at the nucleotide immediately 5' to the polymorphic base. A set of four dideoxynucleotide triphosphate mixtures is formed. Each mixture contains one of the four labeled dideoxynucleotide molecules chemically linked to the fluorescein molecule (ie, ddATP-
F, ddCTP-F, ddGTP-F or ddTTP-F), and three unlabeled dideoxynucleotide triphosphates. In one format, the PCR product is added to beads and the beads are dispensed into two or more tubes. Dispense the chain termination mixture into the tubes and add polymerase to make SBCE reaction tubes. The polymerase extends one base to the 3 'end of the oligo attached to the bead, which is complementary to the base at the polymorphic site. These reaction tubes are analyzed by FlowMetrix, and if a label is found in a particular reaction tube for a particular bead, it indicates the polymorphic base at that site.

Comparison of the present method with the hybridization method demonstrates the utility of the present invention. In SNP analysis by hybridization, 2 beads of 2
The oligo is used to make the material that is read for analysis. In the SCBE method, the same oligo of the same bead is analyzed in two test tubes using two different types of labeled dideoxynucleotides. Although the method is illustrated herein using fluorescein as the reading dye, the method can also be used with biotinylated nucleotides or other appropriately modified nucleotides.

Performing the method wherein the chain terminating nucleotide is a dideoxynucleotide and the primer extension is performed in the presence of one labeled, identified dideoxynucleotide and three different unlabeled dideoxynucleotides Can be. In another embodiment, the chain terminating nucleotide is a dideoxynucleotide, and the primer extension is a first identified dideoxynucleotide labeled with a first detectable label, a second labeled nucleic acid labeled with a second detectable label. Performed in the presence of two identified dideoxynucleotides, a third dideoxynucleotide labeled with a third detectable label, and a fourth dideoxynucleotide labeled with a fourth detectable label; Detection of the presence of the first, second, third, or fourth detectable label in the hybridization product is complementary to a nucleotide selected as the first, second, third, or fourth dideoxynucleotide, respectively. Shows nucleotide characteristics.

FlowMetrix beads are capable of thermal cycling (Ralph McDade, pers. Communication
), Genome scanning can be performed using the SBCE method. In this method, the genome D
NA can be sheared to reduce viscosity or treated with DNase, and recyclable for oligos attached to beads. Due to the tremendous complexity of the template DNA, there may be a need for optimization of hybridization and extension of circulation time. Therefore, the Cot analysis for the beads will be performed practically. SN
The use of these beads and SBCE for P identification and DNA sequencing
It should be clear from the above description.

Accordingly, the present invention provides a method for examining polymorphism of a selected nucleotide in a genomic DNA subjected to a viscosity reducing treatment, comprising: (a) selecting one of the nucleic acid strands at the 5 ′ side of the selected nucleotide Using a first nucleic acid primer containing a region complementary to the section and a second nucleic acid primer complementary to a section of the other nucleic acid strand downstream of the selected nucleotide, between the two primers Amplifying genomic DNA under conditions that specifically amplify the nucleotide region selected in step b) to form a PCR product; and (b) a mobile solid phase that is detectably tagged with the PCR product. A first nucleic acid linked to the support at the 5 'end and comprising a region complementary to a section of one strand of the PCR product immediately 5' to and adjacent to the selected nucleotide; Contacting under hybridization conditions to form a hybridization product; (c) using the hybridization product and a detectably labeled, identified chain terminating nucleotide under primer extension conditions. Performing a primer extension reaction; (d) detecting the presence or absence of a label incorporated in the hybridization product, and detecting the presence of the label indicates that the labeled chain terminating nucleotide is incorporated in the hybridization product. The characteristic of the incorporated labeled chain terminating nucleotide exhibits the characteristic of a nucleotide complementary to the selected nucleotide;
And (e) comparing the characteristics of the selected nucleotide to the non-polymorphic nucleotide, wherein if the characteristics of the selected nucleotide differ from the characteristics of the non-polymorphic nucleotide, a polymorphism of the selected nucleotide is indicated. Providing a method comprising: The PCR product may be in a single-stranded form.

The present invention further provides a method of examining polymorphism of a selected nucleotide in a genomic DNA that has been subjected to a viscosity reduction treatment, the method comprising: (a) selecting a first region having a T7 RNA polymerase promoter; An oligonucleotide containing a second region complementary to a section of one nucleic acid strand immediately 5 ′ to the nucleotide and using as a primer one of the cR
Using T7 RNA polymerase to amplify to NA, and using reverse transcriptase to amplify the other strand complementary to its cRNA strand, under conditions that specifically amplify the nucleotide region between the two primers, Performing amplification of genomic DNA to form an amplification product; (b) the amplification product and a detectably tagged mobile solid support;
Contacting the first oligonucleotide ligated at its terminus under hybridization conditions to form a hybridization product; (c) identifying the hybridization product with a detectably labeled identified oligonucleotide (D) detecting the presence or absence of a label incorporated in the hybridization product, and detecting the presence of the label to determine whether the labeled chain-terminating nucleotide is present. Indicating incorporation into the hybridization product, wherein the characteristic of the incorporated labeled chain terminating nucleotide is characteristic of a nucleotide complementary to the selected nucleotide;
And (e) comparing the characteristics of the selected nucleotide to the non-polymorphic nucleotide, wherein if the characteristics of the selected nucleotide differ from the characteristics of the non-polymorphic nucleotide, the polymorphism of the selected nucleotide is indicated. Providing a method comprising: The labeled chain terminating nucleotide is, for example, 3 ′
It may be a deoxynucleotide, a 3 'deoxyribonucleotide, a thiol nucleotide derivative or a dideoxynucleotide. The amplification product may be in a single-stranded form.

In addition, several primers can be designed and synthesized for placement immediately downstream of the nucleic acid bound to the beads. These can be i) primers used to generate first strand cDNA, and ii) T7 RNA polymerase, along with the set to which it is bound, can be used to generate cRNA. If cRNA is required to make the second strand, a second primer just upstream of the sequence bound to the beads may be used. Even if you have primers away from the bead-oligo, they must not be interfered by binding. These primers can make a FITC label on the cDNA.

The present invention further provides a method for examining polymorphisms of selected nucleotides in a genomic DNA that has been subjected to a viscosity reducing treatment, the method comprising: (a) genomic DNA and a detectably tagged mobile A first primer linked at the 5 'end to the solid support and comprising a first region complementary to a section of one strand of genomic DNA immediately 5' to and adjacent to the selected nucleotide; Is contacted under hybridization conditions to form a specific hybridization product; (b) primer extension using the specific hybridization product and a detectably labeled end-of-chain nucleotide (C) detecting the presence or absence of a label incorporated in the hybridization product, Indicates that the labeled chain terminating nucleotide is incorporated into the hybridization product, wherein the characteristic of the incorporated labeled chain terminating nucleotide is the characteristic of a nucleotide complementary to the selected nucleotide. Showing;
And (d) comparing the characteristics of the selected nucleotide to the non-polymorphic nucleotide, wherein if the characteristics of the selected nucleotide differ from the characteristics of the non-polymorphic nucleotide, a polymorphism of the selected nucleotide is indicated. Providing a method comprising: The DNA may be in single-stranded form. The labeled chain terminating nucleotide may be, for example, a 3 ′ deoxynucleotide, 3 ′ deoxyribonucleotide, thiol nucleotide derivative or dideoxy nucleotide. In such a method, the hybridization time should be long enough to hybridize the genomic DNA with the first primer since genomic DNA was not amplified in this particular embodiment. Thus, as is known in the art for hybridization to genomic DNA, longer hybridization times may be used, for example, 12 hours, 24 hours, 48 hours (eg, Sambrook, et al. Molecular Cloning:
A Laboratory Manual, 2d ed. Cold Spring Harbor Laboratory, Cold Spring H
arbor, New York 1989).

For any of the reactions described herein, the choice is made of a polymerase that has certain temperature conditions for function or that has a specific specificity for nucleotides, such as a polymerase that selectively incorporates dideoxynucleotides. An objective polymerase can be used (see, eg, Sambrook, et al.).
Such polymerases are well known to those of skill in the art, and new polymerases found to be so may be incorporated into the methods taught herein.

The present invention further provides an oligonucleotide ligation assay (OLA) format, ie, a second nucleic acid having a fluorescent label, wherein the genomic DNA, cRNA or PCR product is capable of hybridizing to the first nucleic acid bound to the bead. And the addition of ligase, providing use of the beads in a manner such that the second nucleic acid has a polymorphic base at its 3 'end. Accordingly, the present invention is a method of identifying a selected nucleotide in a first nucleic acid, comprising: (a) a first nucleic acid; and (i) a detectably tagged mobile solid support. 3 'of the test nucleotide, linked at the 5' end, comprising a region complementary to the section of the first nucleic acid immediately 3 'to the selected nucleotide and arranged to base pair with the selected nucleotide. A second nucleic acid terminating in a terminus, and (ii) a fluorescently labeled third nucleic acid comprising a region complementary to a section of the second nucleic acid immediately 5 ′ to and adjacent to the selected nucleotide. Contacting the first nucleic acid and the second nucleic acid under a hybridization condition to form a hybridization product, and the first nucleic acid and the third nucleic acid to form a hybridization product; ) Adding ligase to the hybridization product under ligation reaction conditions; and (c) detecting the presence or absence of a fluorescent label after dissociating the hybridized nucleic acid in the nucleic acid linked to the mobile solid support, The presence indicates a linkage between the labeled third nucleic acid and a second nucleic acid linked to the mobile solid support, and the characteristics of the test nucleotide in the second nucleic acid have been selected. Characterizing nucleotides complementary to the nucleotides and identifying the nucleotides so selected;
Providing a method comprising: This oligonucleotide ligation assay comprises: (a)
The polymorphic base is 5 ′ of either the reporter or the acceptor oligonucleotide
And (b) the polymorphic base is on the 3 ′ side of either the reporter or acceptor oligonucleotide.

The first nucleic acid may be a genomic DNA (for example, one subjected to DNase treatment or viscosity reduction treatment by shearing), an amplified nucleic acid such as a PCR product, an oligonucleotide, a 16s ribosomal RNA, a DNA fragment, RNA Molecule, cDNA molecule, cR
NA molecules, DNA fragments generated by restriction enzymes, size-selective DNA, cross-linked amplification DN
A, 16S RNA, 16S DNA or any other desired nucleic acid. Any selected ligase, such as T4 DNA ligase, may be used. Thermostable ligases will be particularly useful. The outline is Wu and Wallace,
See Genomics 4: 560-569 (1989).

The present invention further provides the use of a degenerate reporter oligonucleotide in OLA reading, preferably wherein six of the bases are selected to be specific for the target nucleic acid,
Includes the use of octamer oligonucleotides in which two of the bases are in variable or wobble or degenerate positions. This degeneracy can be placed at any position on the reporter oligonucleotide, but the preferred positions are 3, 4, 5, and 6
Position. Preferred combinations of variable positions in the selected oligonucleotide may be positions 3 and 6, positions 4 and 5, and positions 3 and 4. Thus, although all possible “6 + dimers” can be synthesized as reagents used in assays, not all possible octamer syntheses are practical.
In addition, non-natural derivatives such as inosine can be used for the reporter oligonucleotide. For example, in the present invention, the reporter oligonucleotide is a vehicle in which the reporter molecule recognizes a hapten (eg, an antibody, avidin, streptavidin)
Base-complementary 8 linked to a reporter molecule or hapten that can be linked by
OLA readings are included. The present invention further provides that the reporter oligonucleotide is a 6-base complementary octamer ("6 + dimer") linked to a reporter molecule or hapten that can be bound by a mediator that recognizes the hapten.
), An OLA reading is included. The two non-complementary bases may be any of the four natural bases or may be non-natural derivatives capable of forming a double helix structure that does not interfere with the helix. These non-complementary bases may preferably be located at positions 3 and 6, or at positions 4 and 5. Unnatural base derivative and / or 6+
The dimer can be a component of a kit used to perform the detection methods described herein.

Further, a dye quencher and a dye acceptor are incorporated into the bound oligo,
A “Taqman” approach can be used to determine whether the polymerase has removed the dye quencher in the repair reaction.

The present invention further provides that although the two nucleic acids hybridize to the same nucleic acid, the ligation step can be omitted and instead the pairing is detected by fluorescence energy transfer between the two nucleic acids hybridized to the sample nucleic acid. Using a hybridization method. The two types of nucleic acids that hybridize are the test base at the 3 'end of the nucleic acid bound to the bead, which is complementary to the polymorphic base, and the energy when the sample is exposed to light of a single wavelength. It is designed to detect the transition and read it as light of a second wavelength. A second reader can be used to detect this second wavelength. Accordingly, the present invention is a method of identifying a selected nucleotide in a first nucleic acid, comprising: (a) a first nucleic acid; and (i) a detectably tagged mobile solid support. Linked at the 5 'end and 3' to the fluorescent label, immediately 3 'of the selected nucleotide
A second nucleic acid comprising a region complementary to a section of the flanking first nucleic acid and terminating at the 3 'end of the test nucleotide arranged to base pair with the selected nucleotide; and (ii) the selected nucleic acid A third nucleic acid comprising a region complementary to a section of the second nucleic acid immediately 5 'to and adjacent to the nucleotide and fluorescently labeled at the 5' end, comprising: a first nucleic acid and a second nucleic acid; To form a hybridization product, and
(C) contacting the nucleic acid with the third nucleic acid under hybridization conditions to form a hybridization product; and (c) a fluorescent label at the 3 ′ end of the second nucleic acid and the 5 ′ end of the third nucleic acid Detecting the presence or absence of fluorescent energy transfer between the fluorescent label and the presence of the fluorescent energy transfer, indicating the hybridization between the test nucleotide and the first nucleic acid;
Identifying a characteristic of the test nucleotide hybridized in the second nucleic acid with a characteristic of the nucleotide complementary to the selected nucleotide, and identifying the nucleotide selected in this manner. The detection of the fluorescence energy transfer (FET) can be performed after the hybridized nucleic acid is dissociated.

[0035] The present invention also provides a method for binding a first nucleic acid, a second nucleic acid, having a 3 'end that is attached to a mobile solid support at the 5' end and is adjacent to a polymorphic base on the second nucleic acid. A third nucleic acid (eg, a first nucleic acid and a third nucleic acid) having a reporter moiety bound complementary to a region adjacent to the base
Nucleic acids together define a polymorphic base and an opposing gap) nucleotides complementary to one of a set of two possible polymorphic bases, a polymerase and a ligase, wherein the polymerase comprises If complementary to the polymorphic base, it can polymerize the nucleotide across the gap, and the ligase can ligate the newly polymerized nucleotide to the third nucleic acid to which the reporter is attached),
And a means for detecting a reporter covalently linked to the beads. In particular, the invention is a method of identifying a selected nucleotide in a first nucleic acid, comprising: (a) a first nucleic acid; and (i) a detectably tagged mobile solid support. A second nucleic acid, linked at the 5 'end, comprising a region complementary to the section of the first nucleic acid immediately 3' to and immediately adjacent to the selected nucleotide; and (ii) Forming a hybridization product of the first nucleic acid and the second nucleic acid with the fluorescently labeled third nucleic acid, which comprises a region complementary to the section of the second nucleic acid immediately 5 ′ and adjacent thereto; And contacting the first nucleic acid and the third nucleic acid under hybridization conditions to form a hybridization product, wherein the first, second, and third nucleic acids form a gap opposite to the selected nucleotide. Regulation (B) adding test nucleotides, polymerase and ligase under polymerization and ligation conditions; and (c) nucleic acids hybridized to nucleic acids linked to the mobile solid support. After dissociation, the presence or absence of a fluorescent label is detected.
And the ligation of the labeled third nucleic acid with a second nucleic acid linked to the mobile solid support, wherein the characteristics of the test nucleotide are complementary to the selected nucleotide. Identifying the nucleotides thus selected and identifying the nucleotides so selected.

The polymerase may preferably be a non-strand-displacement polymerase. Further, it may be a thermostable polymerase. The ligase may be a DNA ligase. Further, it may be a thermostable ligase.

The invention further provides for the modification or endonuclease cleavage of a mismatched base at a polymorphic site in a nucleic acid using an enzyme or chemical, resulting in the loss or modification of the bound reporter and the reporter. A single nucleotide polymorphism detection method is provided that detects a deletion of, or detects a modification, and thus provides a labeling means for identification of the modification. In one example, an end-labeled (such as by FITC) genomic fragment or a labeled (such as by FITC) PCR fragment is hybridized to an oligonucleotide, bound to beads, and the construct is then reconstituted with the mismatched DNA. Recognition and / or restriction enzyme (FITC-labeled recA, m
utS or T7 enzyme) and analyze the addition or deletion of the label. In another example, a chemical that recognizes a single-stranded region of DNA and can modify that region is used to detect this modification.

[0038] Further, any of the methods described herein can be used to quantify the expression of a selected nucleic acid in a sample. Thus, it can be used for differential gene expression, for example, to quantify the expression of a selected gene and compare it to a standard or other reference. For this method, a gene fragment from the region of interest or a region that identifies the gene of interest (or an allele or haplotype or polymorphism) is labeled with a mobile solid support that is detectably labeled. By ligation at the termini, hybridizing a message (eg, RNA, cDNA, cRNA) to the fragment, and performing a primer extension reaction, a ligase reaction, or a hybridization / fluorescence energy transfer reaction such as those described herein. Quantify the fluorescence. The nucleic acid probe can include a region that is complementary to a selected nucleic acid section unique to the nucleic acid. Standards, such as those from a healthy or diseased / diseased subject, or from a particular tissue or organ, or from a particular species, can be used as controls to derive results regarding the amount detected in the sample.

In particular, the present invention relates to a method for detecting a result of an identification reaction to identify a selected nucleotide in a target nucleic acid, comprising: a) a first complementary region and a second complementary region (wherein The two complementary regions are 5 'to the first complementary region and the first complementary region is immediately 3' of the selected nucleotide.
(Including a region complementary to a section of the target nucleic acid on the side and adjacent thereto) and a sample containing the target nucleic acid, the first complementary region of the target oligonucleotide and the target oligonucleotide Contacting under hybridization conditions to form a hybridization product between a target nucleic acid region complementary to the first complementary region to form a first hybridization product; b) said first hybridization product Performing a selected identification reaction comprising a second complementary region of the target oligonucleotide and forming a selectively labeled detection product to characterize the selected nucleotides; c) A nucleic acid covalently linked to the detection product and to a mobile solid support and complementary to a second complementary region of the target oligonucleotide; Isolating the detection product by contacting the capture oligonucleotide containing the sequence under hybridization conditions to form a second hybridization product; and d) the labeled product in the second hybridization product Detecting and / or identifying the label of the detected product, wherein the presence and / or characteristics of the label is characteristic of a selected nucleotide in the target nucleic acid.

Thus, such a basic method is used to isolate a reaction product from a reaction.
Includes the use of capture oligonucleotides attached to a mobile solid support (such as beads). To aid in such isolation, in addition to the first complementarity region, which is a region complementary to the target nucleic acid region, it is 5 'of the first complementarity region and complementary to the nucleotide sequence of the capture oligonucleotide. A "target oligonucleotide" is designed that includes a suitable second complementary region. Thus, before or after the reaction (SBCE or OLA), the capture oligonucleotide can be used in a hybridization reaction to isolate the reacted form of the target oligonucleotide (eg, as a ligation product or a primer extension product). . Thus, as in many other assays, each oligonucleotide (e.g.,
For example, in the present invention, beads need not be specifically synthesized for the first complementary region of the target oligonucleotide).

The invention further encompasses the use of degenerate reporter oligonucleotides in OLA reading, preferably the use of the 6+ dimer described herein. Such a reporter oligonucleotide can be a component of a kit useful for performing the detection methods herein.

The capture oligonucleotide can be designed so that it does not necessarily specifically hybridize, ie, is not sufficiently complementary to the target nucleic acid for specific hybridization to occur. For example, this may include the use of nucleotides not typically found in the target species (such as humans). If the target sequence is sufficiently known, the capture sequence can be selected as a sequence not found in the target sequence. The capture oligonucleotide is sufficiently long to selectively hybridize with the first complementary region of the target oligonucleotide (under selective hybridization conditions, eg, stringent hybridization conditions as known to those of skill in the art). And, unless either the identification reaction performed with the target oligonucleotide, or the hybridization reaction between the capture oligonucleotide and the target oligonucleotide is prevented,
It can be of any desired length. The selected capture oligonucleotide length is also a function of how many different capture oligonucleotides are required for use in any of the selected uses. For example,
Capture oligonucleotides are 8, 10, 12, 15, 18, 20, 21
, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40 or more nucleotides. Preferred lengths are 23, 24, 25, 26
, 27 or 28 nucleotides, about 25 nucleotides. However, other nucleotide lengths can be used. The optimal length for any particular use will depend on the specific nucleic acid composition, as will be known to those skilled in the art.

Advantageously, a bank of several capture oligonucleotides, each linked to a different colored bead, can be created. A bank of complementary regions can be maintained for use in generating target oligonucleotides for any specific target nucleic acid. Therefore, a new target nucleotide required for any detection operation can be easily created using a predetermined set of beads.

The present invention is a method of detecting a reaction product to identify a selected nucleotide in a target nucleic acid, comprising: a) a first complementary region and a second complementary region, wherein the first complementary region The region comprises an oligonucleotide primer and the second complementary region comprises a nucleic acid sequence complementary to the capture oligonucleotide, wherein the oligonucleotide primer is a section of the target nucleic acid immediately 3 ′ to and adjacent to the selected nucleotide. And a sample containing the target nucleic acid.
Contacting under a hybridization condition to form a hybridization product between a first complementary region of the target oligonucleotide and a target nucleic acid region complementary to the first complementary region of the target oligonucleotide, the first hybridization product B) performing a primer extension reaction under primer extension conditions using the first hybridization product and a detectably labeled end-of-chain nucleotide to form a primer extension product. C) combining the primer extension product with a capture oligonucleotide covalently linked to a mobile solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to a second complementary region of the target oligonucleotide. Contact under hybridization conditions, Isolating the primer extension product by forming an isolated second hybridization product; and detecting the presence or absence of a label in the isolated second hybridization product, Indicating that the labeled nucleotide is incorporated into the primer extension product, wherein the characteristics of the identified incorporated labeled nucleotide are indicative of the characteristics of a nucleotide that is complementary to the selected nucleotide and thus selected in the target nucleic acid. Identifying the identified nucleotide.

In a typical assay, the target oligonucleotide comprises a second complementary region at the 5 ′ end that is subsequently capable of hybridizing to a complementary capture oligonucleotide linked to a mobile solid support, Is designed to include a first complementary region that is complementary to the target nucleic acid region immediately 3 ′ to the polymorphic base. This 3 'base is
Depending on the assay being performed, terminates in a nucleotide chosen for the polymorphic base. For example, the 3 'base of the target oligonucleotide may end with a nucleotide 5' to the polymorphic base, or may end with a base corresponding to the polymorphic base. The present invention further provides an oligonucleotide ligation assay (OLA) format, ie, a method in which a genomic DNA, cRNA or PCR product has a first complementary region complementary to a section of the target nucleic acid immediately 3 ′ to the selected nucleotide. Hybridization with the oligonucleotide, followed by the inclusion of a reporter oligonucleotide with a fluorescent label, followed by the addition of ligase, provides for the use of beads in a manner where the target oligonucleotide has a polymorphic base at the 3 'end. For a typical OLA reaction with a capture read, the reagents include two complementary regions (the first complementary region of the target oligo is complementary to the region immediately adjacent to the single nucleotide polymorphism being analyzed). And the second complementary region of the target oligonucleotide is complementary to the capture oligonucleotide).
A capture oligonucleotide covalently linked to a mobile solid support; a reporter oligonucleotide that covers the SNP and is complementary to the region containing the reading means and the 3 'base on the strand opposite the SNP position; on the reporter opposite the SNP If the bases are complementary, a ligase capable of linking the reporter to the target may be included. In one embodiment of the method, the ligation reaction is then added to a mobile solid support to which the capture-oligonucleotide is attached, allowing hybridization of the second complementary region to the beads under standard hybridization conditions. Let it. The reporter reading can be performed using a Luminex LX100 machine.

An advantage of this system is that the number of bead sets required to analyze many different SNPs is reduced, ie, given 100 bead colors, 100 capture oligos Only nucleotides are synthesized and can be used many times in different wells.

Accordingly, the present invention provides a method for identifying a selected nucleotide in a target nucleic acid by detecting the result of an identification reaction (OLA), comprising: a) (i) a first complementary region and a second complementary region (Where the first complementary region comprises a region complementary to the section of the target nucleic acid immediately 3 ′ to and adjacent to the selected nucleotide, and the second complementary region comprises a region complementary to the capture oligonucleotide. And (ii) a fluorescently-labeled reporter oligonucleotide comprising a region complementary to a section of the target nucleic acid immediately 5 ′ to and adjacent to the selected nucleotide. Specifically hybridizing between a first complementary region of the target oligonucleotide and a target nucleic acid region complementary to the first complementary region of the target oligonucleotide And hybridizing with a sample containing the target nucleic acid under hybridization conditions that specifically hybridize between the reporter oligonucleotide and a section of the target nucleic acid that is complementary to the reporter oligonucleotide. Forming a first hybridization product defining an opposite gap; b) adding the identified test nucleotide, polymerase and ligase under polymerization and ligation conditions to form a labeled product; c). Dissociating the hybridized nucleic acid; d) the labeled product and a capture oligonucleotide covalently linked to a mobile solid support, wherein the capture oligonucleotide binds to a second complementary region of the target oligonucleotide. Contains complementary nucleic acid sequence Isolating the labeled product by contacting under hybridization conditions to form a second hybridization product; and e) detecting the presence or absence of the label in the second hybridization product. Wherein the presence of the label indicates polymerization of the identified test nucleotide with the target oligonucleotide, and ligation of the labeled reporter oligonucleotide with the polymerized target oligonucleotide, wherein the identified test nucleotide is characteristic. Characterizing nucleotides complementary to the selected nucleotides and thus identifying the selected nucleotides in the target nucleic acid.

The target nucleic acid is genomic DNA, oligonucleotide, 1
It may be 6s ribosomal RNA, 16S DNA, PCR product, DNA fragment, DNA fragment generated by restriction enzyme, size selected DNA, cross-amplified DNA, RNA molecule, cDNA molecule or cRNA molecule.

The present invention further provides a method of examining polymorphism of a selected nucleotide in a genomic DNA subjected to a viscosity reducing treatment, comprising: a) adding a section of one nucleic acid strand 5 ′ to the selected nucleotide; Using a first nucleic acid primer containing a complementary region and a second nucleic acid primer complementary to a section of the other nucleic acid strand downstream of the selected nucleotide, selecting between the two primers Amplifying the genomic DNA under conditions that specifically amplify the isolated nucleotide region to form a PCR product; b) the PCR product and a first complementary region and a second complementary region (wherein One complementary region is immediately 5 'of the selected nucleotide and is complementary to a section of one strand of the PCR product adjacent thereto). Contacting under hybridization conditions to form a first hybridization product; c) using the first hybridization product and a detectably labeled chain terminating nucleotide, Performing a primer extension reaction under primer extension conditions to form a primer extension product; d) a capture oligonucleotide covalently linked to the mobile solid support, wherein the capture oligonucleotide is a target Isolating the primer extension product by contacting the oligonucleotide with a second complementary region of the oligonucleotide (comprising a nucleic acid sequence complementary to the second complementary region) under hybridization conditions to form an isolated second hybridization product. E) adding to the second hybridization product Detecting the presence or absence of the incorporated label, the presence of the label indicates that the labeled chain terminating nucleotide has been incorporated into the primer extension product, and the characteristics of the incorporated labeled chain terminating nucleotide are: Exhibit characteristics of nucleotides complementary to the selected nucleotides; and f) comparing the characteristics of the selected nucleotides to non-polymorphic nucleotides, wherein the characteristics of the selected nucleotides are the same as those of the non-polymorphic nucleotides. Differing, the polymorphism of the selected nucleotide is indicated.

The present invention further provides a method for examining polymorphism of a selected nucleotide in a genomic DNA subjected to a viscosity reducing treatment, comprising: a) a first region having a T7 RNA polymerase promoter; An oligonucleotide containing a second region complementary to a section of one of the nucleic acid strands immediately 5 ′ to the first strand is used as a primer, and
A, using T7 RNA polymerase to amplify to A and reverse transcriptase to amplify the other strand complementary to its cRNA strand, under conditions that specifically amplify the nucleotide region between the two primers Amplifying the DNA to form an amplification product; b) the amplification product and a first complementary region and a second complementary region, wherein the first complementary region is immediately 5 ′ to the selected nucleotide. And is complementary to a section of one strand of the PCR product adjacent thereto and the second complementary region is 5% of the first complementary region.
Contacting with a target oligonucleotide comprising the first hybridization product under hybridization conditions to form a first hybridization product; c) the first hybridization product is detectably labeled. Performing a primer extension reaction under primer extension conditions using the identified chain terminating nucleotide to form a primer extension product; d) a capture covalently bound to the primer extension product and to a mobile solid support. Contacting the oligonucleotide (where the capture oligonucleotide comprises a nucleic acid sequence complementary to the second complementary region of the target oligonucleotide) under hybridization conditions to isolate the isolated second hybridization product. Isolating the primer extension product by forming e) detecting the presence or absence of a label incorporated into the second hybridization product, the presence of the label indicating that the labeled chain terminating nucleotide is incorporated into the primer extension product, and The characteristics of the labeled chain-terminating nucleotide exhibit characteristics of nucleotides complementary to the selected nucleotide; and f) comparing the characteristics of the selected nucleotide to non-polymorphic nucleotides and determining the characteristics of the selected nucleotide. If the characteristic differs from that of the non-polymorphic nucleotide, indicating a polymorphism of the selected nucleotide.

The labeled chain terminating nucleotide may be, for example, a 3 ′ deoxynucleotide, 3 ′ deoxyribonucleotide, thiol nucleotide derivative or dideoxy nucleotide. The amplification product may be in a single-stranded form. In addition, several primers immediately downstream of the target oligonucleotide can be designed and synthesized.

The present invention further provides a method for examining the polymorphism of selected nucleotides in a genomic DNA that has been subjected to a viscosity reducing treatment, the method comprising: a) genomic DNA, a first complementary region and a second complementary region ( Here, the first complementary region is complementary to a section of one strand of the PCR product immediately 5 'to and adjacent to the selected nucleotide, and the second complementary region is complementary to the first complementary region. Contacting with a target oligonucleotide comprising a 5 ') oligonucleotide under hybridization conditions to form a specific first hybridization product; b) said specific first hybridization product; Performing a primer extension reaction under conditions of primer extension using an identified chain terminating nucleotide that is detectably labeled; c) said primer extension And a capture oligonucleotide covalently linked to a mobile solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to the second complementary region of the target oligonucleotide. Isolating the primer extension product by forming an isolated second hybridization product by contacting with d); d) detecting the presence or absence of a label incorporated in the second hybridization product, Indicates that the labeled chain terminating nucleotide is incorporated into the hybridization product, wherein the characteristic of the incorporated labeled chain terminating nucleotide is the characteristic of a nucleotide complementary to the selected nucleotide. And e) determining the characteristics of the selected nucleotides by a non-polymorphic nucleic acid. Comparing the characteristics of the selected nucleotide to those of the non-polymorphic nucleotide as compared to an otide, indicating a polymorphism of the selected nucleotide.

[0053] The DNA may be in single-stranded form. The labeled chain terminating nucleotide is, for example,
It may be a 3 'deoxynucleotide, 3' deoxyribonucleotide, thiol nucleotide derivative or dideoxynucleotide. In such a method, the genomic DNA has not been amplified in this particular embodiment, so the hybridization time must be long enough to allow the first primer to hybridize to the genomic DNA. Thus, as is known in the art for hybridization to genomic DNA, for example, 12 hours, 24 hours,
Longer hybridization times, such as 48 hours, can be used (see, eg, Sambrook, et al.).

In the reaction using ligase, any selected ligase such as T4 DNA ligase may be used. Thermostable ligases will be particularly useful. The outline is Wu and
See Wallace, Genomics 4: 560-569 (1989).

The present invention further provides that the two nucleic acids hybridize to the sample nucleic acid, but the ligation step can be omitted, and instead the pairing is detected by fluorescence energy transfer between the two nucleic acids hybridized with the sample nucleic acid. Using a hybridization method. The two hybridizing nucleic acids have a test base at the 3 'end of the target oligonucleotide, which is complementary to the polymorphic base, and has a low energy when the sample is exposed to a single wavelength of light. It is designed so that the transition can be detected and read as light of a second wavelength. For detection of this second wavelength, a second reader can be used. Accordingly, the invention is a method of identifying selected nucleotides in a target nucleic acid, comprising: a) a target nucleic acid; i. A first complementary region and a second complementary region, wherein the first complementary region is complementary to a section of the first nucleic acid immediately 5 ′ to the selected nucleotide and the target oligonucleotide is selected A target oligonucleotide terminated at the 3 'end of the identified test nucleotide positioned to base pair with the second nucleotide and the second complementary region is 5' to the first complementary region). And ii. A fluorescently-labeled reporter oligonucleotide comprising a region complementary to the section of the target nucleic acid immediately 3 ′ to and adjacent to the selected nucleotide, hybridizing the target nucleic acid and the target oligonucleotide; and Contacting the target nucleic acid with a reporter oligonucleotide under hybridization conditions to form a first hybridization product; b) adding a ligase to the first hybridization product under ligation conditions; c. A) the first hybridization product and a capture oligonucleotide covalently linked to a mobile solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to a second complementary region of the target oligonucleotide; And the hybridization Isolating the first hybridization product by contacting under hybridization conditions to form an isolated second hybridization product; and, in the second hybridization product, After dissociation, the presence or absence of a fluorescent label is detected, and the presence of the label indicates the link between the labeled reporter oligonucleotide and the target oligonucleotide, and the characteristics of the test nucleotide in the target oligonucleotide are selected nucleotides. Characterizing nucleotides complementary to and identifying the nucleotides so selected. The detection of the fluorescent energy transfer can be performed after the hybridized nucleic acid is dissociated.

The present invention further provides a method of identifying selected nucleotides in a target nucleic acid, comprising: a) a target nucleic acid; i. Linked to the fluorescent label at the 3 'end, comprising a first complementary region complementary to the section of the target nucleic acid immediately 3' to the selected nucleotide, and arranged to base pair with the selected nucleotide. A labeled oligonucleotide terminating at the 3 'end of the test nucleotide and having a second complementary region 5' to the first complementary region;
And ii. A reporter oligonucleotide that is fluorescently labeled at the 5 ′ end and contains a region complementary to a section of the target nucleic acid immediately 5 ′ to and adjacent to the selected nucleotide is hybridized with the target nucleic acid and the target oligonucleotide. And contacting the target nucleic acid with a reporter oligonucleotide under hybridization conditions to form a first hybridization product; b) attaching the first hybridization product to a mobile solid support. A covalently attached capture oligonucleotide, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to the second complementary region of the target oligonucleotide, is contacted under hybridization conditions with the isolated oligonucleotide. 2 hybridization products Isolating the first hybridization product by fluorescence; and, in the second hybridization product, a fluorescent label at the 3 'end of the target oligonucleotide and a fluorescence at the 5' end of the reporter oligonucleotide. Detecting the presence or absence of a fluorescent energy transfer between the label and the presence of the fluorescent energy transfer indicates hybridization of the identified test nucleotide to the target nucleic acid and characteristics of the hybridized test nucleotide in the target oligonucleotide. Characterizing nucleotides that are complementary to the selected nucleotides and identifying the nucleotides so selected.

The present invention also provides a target oligonucleotide comprising: a first complementary region and a second complementary region 5 ′ to the first complementary region (where the first complementary region is a target oligonucleotide). Complementary to a section of the target nucleic acid having a 3 ′ end immediately 5 ′ to and adjacent to the polymorphic base on the nucleic acid); 3 ′ to and directly adjacent to the polymorphic base of the target nucleic acid A reporter oligonucleotide having a reporter moiety complementary to the region bound; a target oligonucleotide and a reporter oligonucleotide that together define an opposing gap for the polymorphic base; shared with a mobile solid support (such as polystyrene-divinylbenzene beads). Bound capture oligonucleotide (where the capture oligonucleotide is complementary to the second complementary region of the target oligonucleotide) Nucleotides complementary to one of a set of two possible polymorphic bases; a polymerase, and a ligase (where the polymerase is a gap if the nucleotide is complementary to the polymorphic base). A ligase can link newly polymerized nucleotides to a reporter oligonucleotide); and a kit comprising one or more means for detecting the reporter covalently attached to the beads. And a method for determining the sequence of a polymorphic base in a target nucleic acid. Further, the invention is a method of identifying selected nucleotides in a target nucleic acid, comprising: a) a target nucleic acid; i. A first complementary region and a second complementary region 5 'to the first complementary region, wherein the first complementary region is immediately 3' to the selected nucleotide and directly adjacent to the section of the target nucleic acid. A target oligonucleotide, which is complementary), and ii. A fluorescently labeled reporter oligonucleotide, comprising a region complementary to the section of the second nucleic acid immediately 5 ′ to and adjacent to the selected nucleotide, is used to bind the target nucleic acid and the hybridization product to the target oligonucleotide. Forming and contacting the target nucleic acid and the reporter oligonucleotide under hybridization conditions to form a hybridization product, wherein the target nucleic acid, the target oligonucleotide and the reporter oligonucleotide define an opposing gap of the selected nucleotide. Forming a hybridization product; b) adding the identified test nucleotide, polymerase and ligase under polymerization and ligation conditions; c) covalently binding the first hybridization product to a mobile solid support. A contacting capture oligonucleotide, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to the second complementary region of the target oligonucleotide, under hybridization conditions. Isolating the first hybridization product by forming a hybridization product of the above; and d) detecting the presence or absence of a fluorescent label in the second hybridization product after dissociating the hybridized nucleic acid Wherein the presence of the label indicates polymerization of the test nucleic acid with the target oligonucleotide, and ligation of the labeled reporter oligonucleotide with the target oligonucleotide linked to the mobile solid support,
Identifying the nucleotides that are complementary to the selected nucleotides and identifying the nucleotides so selected.

The polymerase may preferably be a non-strand displacement polymerase. Further, it may be a thermostable polymerase. The ligase may be a DNA ligase. Further, it may be a thermostable ligase.

In addition, any of the methods described herein can be used for quantifying the expression of a selected nucleic acid in a sample. Thus, it can be used, for example, for differential gene expression, where the expression of a selected gene is quantified and compared to a standard or other reference. For this method, a gene fragment from the region of interest or a region that identifies the gene of interest (or allele or haplotype or polymorphism) is selected for use as the first complementary region of the target oligonucleotide. Then, a message (eg, RNA, cDNA, cRNA) is hybridized to the target oligonucleotide and fluorescence is obtained by performing a primer extension reaction, a ligase reaction, or a hybridization / fluorescence energy transfer reaction such as those described herein. Quantify. The reaction product is supplemented with a corresponding capture oligonucleotide (complementary to the second complementary region used in the target oligonucleotide) linked to a mobile solid support. The first complementary region of the target oligonucleotide can include a region that is complementary to a selected nucleic acid section that is unique to the nucleic acid. Standards, such as those from a healthy or diseased / diseased subject, or from a particular tissue or organ, or from a particular species, can be used as controls to derive results regarding the amount detected in the sample.

Accordingly, the invention is a method of quantifying the expression of a selected nucleic acid in a sample, comprising: a) (i) a message nucleic acid isolated from a selected source; and (ii) a first complementary region. And contacting with a target oligonucleotide comprising a second complementary region 5 'to the first complementary region, wherein the first complementary region comprises a region complementary to a section of the selected nucleic acid. B) using the first hybridization product to perform a selected identification reaction that includes the second complementary region of the target oligonucleotide and that can form a selectively labeled detection product; Characterizing the nucleotides; c) a capture oligonucleotide covalently linked to the detection product and to a mobile solid support and comprising a nucleic acid sequence complementary to a second complementary region of a target oligonucleotide. Isolating the detection product by contacting it with a reotide under hybridization conditions to form an isolated hybridization product; and d) quantifying the fluorescence in the isolated hybridization product, Wherein the amount of fluorescence indicates the amount of the selected nucleic acid in the sample.

In any of these methods described herein, the sample may be, for example, blood, red blood cells or white blood cells, bone marrow, liver, kidney, skin, heart, lung, spleen, pancreas, gallbladder, muscle, nerve cells, neurons, progenitors. It may be any body sample containing messages such as cells, organ tissues such as ovaries, testes, uterus, glands and / or cells.

Also provided is a single base polymorphism detection kit, wherein the kit is detectably tagged, a mobile solid support such as polystyrene-divinylbenzene beads, and a 3 ′ deoxynucleotide. , M, 3 'deoxyribonucleotide,
It comprises one to four modified (chain terminated) nucleotides such as thiol derivatives or dideoxynucleotides. The kit may further include a polymerase, particularly a polymerase that selectively incorporates the modified nucleotides. Further, the kit may include a ligase. The kit may also include one or more fluorescent labels for labeling nucleic acids. For genomic DNA applications, the kit may further include a DNase that reduces the viscosity of the DNA. The kit may further comprise a combinatorial array of dinucleotides and / or a combinatorial collection of trinucleotides.

The following documents provide information on various technologies. PCT publication WO9714028 (Luminex Corp.) Australian patent AU9723205 (based on WO9735033 (97/09/25)) (Molecular T
ool Inc.) European Patent Publication EP754240 (based on WO9521271) (Molecular Tool Inc.) European Patent Publication EP736107 (based on WO9517524) (Molecular Tool Inc.) U.S. Pat. No. 5,610,287 (97/03/11) (Molecular Tool Inc .) European Patent Publication EP726905 (based on WO9512607) (Molecular Tool Inc.) US Patent No. 5,518,900 (94/07/21) (Molecular Tool Inc.) European Patent Publication EP576558 (based on WO9215712) (Molecular Tool Inc.) Throughout this application, various publications are referenced. These publications are deemed to be part of the present specification by reference.

Although the invention has been described with respect to certain specific embodiments, it is contemplated that many modifications and changes may be made by those skilled in the art without departing from the spirit of the invention. Therefore,
All such modifications and variations that fall within the true spirit and scope of the invention are intended to be covered by the appended claims.

[Procedure amendment]

[Submission date] April 25, 2001 (2001. 4.25)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claim 70

[Correction method] Change

[Correction contents]

[Procedure amendment 2]

[Document name to be amended] Statement

[Correction target item name] 0034

[Correction method] Change

[Correction contents]

The present invention further provides that although the two nucleic acids hybridize to the same nucleic acid, the ligation step can be omitted and instead the pairing is detected by fluorescence energy transfer between the two nucleic acids hybridized to the sample nucleic acid. Using a hybridization method. The two types of nucleic acids that hybridize are the test base at the 3 'end of the nucleic acid bound to the bead, which is complementary to the polymorphic base, and the energy when the sample is exposed to light of a single wavelength. It is designed to detect the transition and read it as light of a second wavelength. A second reader can be used to detect this second wavelength. Accordingly, the present invention is a method of identifying a selected nucleotide in a first nucleic acid, comprising: (a) a first nucleic acid; and (i) a detectably tagged mobile solid support. Linked at the 5 'end and 3' to the fluorescent label, immediately 3 'of the selected nucleotide
A second nucleic acid comprising a region complementary to a section of the flanking first nucleic acid and terminating at the 3 'end of the test nucleotide arranged to base pair with the selected nucleotide; and (ii) the selected nucleic acid A third nucleic acid comprising a region complementary to a section of the second nucleic acid immediately 5 'to and adjacent to the nucleotide and fluorescently labeled at the 5' end, comprising: a first nucleic acid and a second nucleic acid; To form a hybridization product, and
Contacting the nucleic acid with the third nucleic acid under hybridization conditions to form a hybridization product; and ( b ) a fluorescent label at the 3 ′ end of the second nucleic acid and the 5 ′ end of the third nucleic acid Detecting the presence or absence of fluorescent energy transfer between the fluorescent label and the presence of the fluorescent energy transfer, indicating the hybridization between the test nucleotide and the first nucleic acid;
Identifying a characteristic of the test nucleotide hybridized in the second nucleic acid with a characteristic of the nucleotide complementary to the selected nucleotide, and identifying the nucleotide selected in this manner. The detection of the fluorescence energy transfer (FET) can be performed after the hybridized nucleic acid is dissociated.

[Procedure amendment 3]

[Document name to be amended] Statement

[Correction target item name] 0044

[Correction method] Change

[Correction contents]

The present invention is a method of detecting a reaction product to identify a selected nucleotide in a target nucleic acid, comprising: a) a first complementary region and a second complementary region, wherein the first complementary region The region comprises an oligonucleotide primer and the second complementary region comprises a nucleic acid sequence complementary to the capture oligonucleotide, wherein the oligonucleotide primer is a section of the target nucleic acid immediately 3 ′ to and adjacent to the selected nucleotide. And a sample containing the target nucleic acid.
Contacting under a hybridization condition to form a hybridization product between a first complementary region of the target oligonucleotide and a target nucleic acid region complementary to the first complementary region of the target oligonucleotide, the first hybridization product B) performing a primer extension reaction under primer extension conditions using the first hybridization product and a detectably labeled end-of-chain nucleotide to form a primer extension product. C) combining the primer extension product with a capture oligonucleotide covalently linked to a mobile solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to a second complementary region of the target oligonucleotide. Contact under hybridization conditions, By forming a second hybridization product, which is, that the isolation of the primer extension product; and d) detecting the presence or absence of label in the isolated second hybridization product, the presence of the label Indicating that the labeled nucleotide is incorporated into the primer extension product, wherein the characteristics of the identified incorporated labeled nucleotide are characteristic of nucleotides complementary to the selected nucleotide, and thus the target nucleic acid Identifying the selected nucleotide in.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0055

[Correction method] Change

[Correction contents]

The present invention further provides that the two nucleic acids hybridize to the sample nucleic acid, but the ligation step can be omitted, and instead the pairing is detected by fluorescence energy transfer between the two nucleic acids hybridized with the sample nucleic acid. Using a hybridization method. The two hybridizing nucleic acids have a test base at the 3 'end of the target oligonucleotide, which is complementary to the polymorphic base, and has a low energy when the sample is exposed to a single wavelength of light. It is designed so that the transition can be detected and read as light of a second wavelength. For detection of this second wavelength, a second reader can be used. Accordingly, the invention is a method of identifying selected nucleotides in a target nucleic acid, comprising: a) a target nucleic acid; i. A first complementary region and a second complementary region, wherein the first complementary region is complementary to a section of the first nucleic acid immediately 5 ′ to the selected nucleotide and the target oligonucleotide is selected A target oligonucleotide terminated at the 3 'end of the identified test nucleotide positioned to base pair with the second nucleotide and the second complementary region is 5' to the first complementary region). And ii. A fluorescently-labeled reporter oligonucleotide comprising a region complementary to the section of the target nucleic acid immediately 3 ′ to and adjacent to the selected nucleotide, hybridizing the target nucleic acid and the target oligonucleotide; and Contacting the target nucleic acid with a reporter oligonucleotide under hybridization conditions to form a first hybridization product; b) adding a ligase to the first hybridization product under ligation conditions; c. A) the first hybridization product and a capture oligonucleotide covalently linked to a mobile solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to a second complementary region of the target oligonucleotide; And the hybridization Isolating said first hybridization product by contacting under contact conditions to form an isolated second hybridization product; and d) hybridizing at said second hybridization product. Detecting the presence or absence of a fluorescent label after dissociating the nucleic acid, the presence of the label indicates the link between the labeled reporter oligonucleotide and the target oligonucleotide, and the characteristics of the test nucleotide in the target oligonucleotide are selected. Characterizing the nucleotides complementary to the nucleotides identified and identifying the nucleotides selected in this manner. The detection of the fluorescent energy transfer can be performed after the hybridized nucleic acid is dissociated.

[Procedure amendment 5]

[Document name to be amended] Statement

[Correction target item name] 0056

[Correction method] Change

[Correction contents]

The present invention further provides a method of identifying selected nucleotides in a target nucleic acid, comprising: a) a target nucleic acid; i. Linked to the fluorescent label at the 3 'end, comprising a first complementary region complementary to the section of the target nucleic acid immediately 3' to the selected nucleotide, and arranged to base pair with the selected nucleotide. A labeled oligonucleotide terminating at the 3 'end of the test nucleotide and having a second complementary region 5' to the first complementary region;
And ii. A reporter oligonucleotide that is fluorescently labeled at the 5 ′ end and contains a region complementary to a section of the target nucleic acid immediately 5 ′ to and adjacent to the selected nucleotide is hybridized with the target nucleic acid and the target oligonucleotide. And contacting the target nucleic acid with a reporter oligonucleotide under hybridization conditions to form a first hybridization product; b) attaching the first hybridization product to a mobile solid support. A covalently attached capture oligonucleotide, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to the second complementary region of the target oligonucleotide, is contacted under hybridization conditions with the isolated oligonucleotide. 2 hybridization products First thing the hybridization product is isolated by making; in and c) the second hybridization product, the 5 'end of the fluorescent label and the reporter oligonucleotide in 3' end of the target oligonucleotide Detecting the presence or absence of fluorescent energy transfer between a fluorescent label and the presence of the fluorescent energy transfer indicates hybridization between the identified test nucleotide and the target nucleic acid, and the hybridized test nucleotide in the target oligonucleotide The characteristic of the nucleotide complementary to the selected nucleotide,
Identifying the nucleotides selected in this manner.

──────────────────────────────────────────────────続 き Continuation of the front page (31) Priority claim number 60/100, 703 (32) Priority date September 17, 1998 (September 17, 1998) (33) Priority claim country United States (US) ( 31) Priority claim number 60 / 108,018 (32) Priority date November 12, 1998 (November 12, 1998) (33) Priority claim country United States (US) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ , TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK , MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Glaxo Wellcome House, Berkeley Avenue Greenfield, Middlesex UB6 0NN, Great Brita in (72) Inventors Weiner, Michael Phillip A United States of America, North Carolina 27709-3398 Research Triangle Park, P.O.Box 13398, Five Moore Drive, Glaxo Welcome Inc. F-Terms (reference) 4B024 AA01 AA11 AA13 CA01 CA04 CA09 CA11 CA20 DA03 HA08 HA11 HA14 4B063 QA01 QA12 QA17 QA18 QA19 QQ02 QQ03 QQ08 QQ41 QQ42 QQ52 QQ54 QQ58 QQ60 QR08 QR14 QR31 QR32 QR35 QR62 QR66 QR82 QR83 QS25 QS34 QS36 QX02

Claims (73)

[Claims] 1. A method for identifying a selected nucleotide in a first nucleic acid comprising the steps of: a) 5 ′ on a first solid phase and a detectably tagged mobile solid support.
Ligation at the end, the first 3 'immediately adjacent and adjacent to the selected nucleotide
Contacting a nucleic acid primer comprising a region complementary to a section of the nucleic acid under hybridization conditions to cause the first nucleic acid and the nucleic acid primer to form a hybridization product; b) contacting the hybridization product with a detectable nucleic acid; Performing a primer extension reaction under primer extension conditions using the identified chain terminating nucleotide labeled as above; and c) detecting the presence or absence of a label incorporated in the hybridization product, and detecting the presence of the label. Indicating that the labeled nucleotide is incorporated into the hybridization product, wherein the characteristics of the incorporated labeled nucleotide indicate characteristics of a nucleotide complementary to the selected nucleotide, and thus the first Identify selected nucleotides in nucleic acids When; method comprising. 2. The method according to claim 1, wherein the first nucleic acid is an oligonucleotide, a 16s ribosomal RNA, a PCR product, a DNA fragment, an RNA molecule, a cDNA molecule or a cRNA molecule, and the nucleic acid primer is an oligonucleotide, a PCR product, a DNA fragment, The method according to claim 1, which is an RNA molecule, a cDNA molecule or a cRNA molecule. 3. The method of claim 1, wherein the labeled chain terminating nucleotide is a 3 ′ deoxynucleotide, 3 ′ deoxyribonucleotide, thiol nucleotide derivative or dideoxy nucleotide. 4. The chain terminating nucleotide is a dideoxynucleotide,
2. The method of claim 1, wherein the primer extension is performed in the presence of one labeled dideoxynucleotide identified and three different unlabeled dideoxynucleotides. 5. The chain terminating nucleotide is a dideoxynucleotide,
The primer extension comprises: a first identified dideoxynucleotide labeled with a first detectable label; a second identified dideoxynucleotide labeled with a second detectable label; a third detectable label. Performed in the presence of a third identified dideoxynucleotide labeled with a novel label, and a fourth identified dideoxynucleotide labeled with a fourth detectable label, and detectable in the hybridization product Detection of the presence of the first, second, third or fourth detectable label is determined by determining whether the nucleotide complementary to the selected nucleotide is the first, second or third nucleotide, respectively.
, Or a third or fourth dideoxynucleotide.
The method described in. 6. The method of claim 1, wherein said mobile solid support is a bead. 7. The method of claim 4, wherein said beads are polystyrene-divinylbenzene. 8. The method of claim 1, wherein the mobile solid support is tagged so as to be detectable with a dye, a radiolabel, or a magnetic tag. 9. The method according to claim 1, wherein said first nucleic acid is an amplification product. 10. The method according to claim 1, wherein said first nucleic acid is a PCR product. 11. The method of claim 1, wherein the detection is performed by passing the movable solid support over a laser detection device capable of detecting / identifying a detectable tag. 12. The detection is carried out by fixing the movable solid support on a two-dimensional surface and passing a laser detection device capable of detecting / identifying a detectable tag on the solid support. 2. The method according to 1. 13. A method for examining polymorphism of a selected nucleotide in a genomic DNA subjected to a viscosity reduction treatment, comprising: a) complementary to a section of one nucleic acid strand on the 5 ′ side of the selected nucleotide. Selected between the two primers using a first nucleic acid primer containing a unique region and a second nucleic acid primer complementary to a section of the other nucleic acid strand downstream of the selected nucleotide. Amplifying the genomic DNA under conditions that specifically amplify the nucleotide region to form a PCR product; b) the PCR product, a detectably tagged mobile solid support and 5 ′ Hybridizing with a first nucleic acid that is ligated at the terminus and contains a region complementary to a section of one strand of the PCR product immediately 5 ′ to and adjacent to the selected nucleotide Contacting under hybridization conditions to form a hybridization product; c) primer extension under primer extension conditions using said hybridization product and a detectably labeled, identified chain terminating nucleotide. Performing a reaction; d) detecting the presence or absence of a label incorporated in the hybridization product, and the presence of the label indicating that the labeled chain-terminating nucleotide is incorporated in the hybridization product; Wherein the characteristics of the labeled, labeled chain-terminating nucleotides exhibit characteristics of nucleotides complementary to the selected nucleotides; and e) comparing the characteristics of the selected nucleotides to non-polymorphic nucleotides. If the characteristics of the nucleotide are different from those of the non-polymorphic nucleotide, The method comprising; that polymorphic nucleotides are shown. 14. The method of claim 13, wherein said labeled chain terminating nucleotide is a 3 'deoxynucleotide, 3' deoxyribonucleotide, thiol nucleotide derivative or dideoxy nucleotide. 15. The method of claim 13, wherein the DNA has been sheared to reduce viscosity. 16. The method of claim 13, wherein the DNA has been treated with DNase to reduce viscosity. 17. A method for examining polymorphisms of selected nucleotides in a genomic DNA subjected to a viscosity reduction treatment, comprising: a) a first region having a T7 RNA polymerase promoter and 5 min. An oligonucleotide containing a second region complementary to a section of one nucleic acid strand on the 'side is used as a primer, and one of the strands is cRN
A, using T7 RNA polymerase to amplify to A and reverse transcriptase to amplify the other strand complementary to its cRNA strand, under conditions that specifically amplify the nucleotide region between the two primers Amplifying the DNA to form an amplification product; b) combining said amplification product with a detectably tagged mobile solid support;
Contacting the first oligonucleotide ligated at the terminus with hybridization conditions under hybridization conditions to form a hybridization product; c) the hybridization product is identified as detectably labeled Performing a primer extension reaction under primer extension conditions using a chain terminating nucleotide; d) detecting the presence or absence of a label incorporated in the hybridization product; Indicating that it is incorporated into the hybridization product, wherein the characteristic of the incorporated labeled chain-terminating nucleotide is characteristic of a nucleotide complementary to the selected nucleotide; and e) the characteristic of the selected nucleotide. Compared to non-polymorphic nucleotides, the selected Wherein the characteristics of the leotide differ from the characteristics of the non-polymorphic nucleotide, indicating a polymorphism of the selected nucleotide. 18. The method of claim 17, wherein said labeled chain terminating nucleotide is a 3 'deoxynucleotide, 3' deoxyribonucleotide, thiol nucleotide derivative or dideoxy nucleotide. 19. A method for examining the polymorphism of selected nucleotides in a genomic DNA that has been subjected to a viscosity reduction treatment, comprising: a) a genomic DNA and a detectably tagged mobile solid support. And 5
A first primer ligated at the terminus and containing a first region complementary to a section of one strand of genomic DNA immediately 5 'to and adjacent to the selected nucleotide is contacted under hybridization conditions. B) performing a primer extension reaction under primer extension conditions using said specific hybridization product and a detectably labeled end-of-chain nucleotide. C) detecting the presence or absence of a label incorporated into the hybridization product, wherein the presence of the label indicates that the labeled chain-terminating nucleotide is incorporated into the hybridization product; The characteristics of the terminating nucleotide at the end of the Exhibiting the characteristics of the otide; and d) comparing the characteristics of the selected nucleotide to the non-polymorphic nucleotide, and if the characteristics of the selected nucleotide differ from the characteristics of the non-polymorphic nucleotide, Exhibiting polymorphism; 20. The method of claim 19, wherein the labeled chain terminating nucleotide is a 3 'deoxy nucleotide, a 3' deoxy ribonucleotide, a thiol nucleotide derivative or a dideoxy nucleotide. 21. A method for identifying a selected nucleotide in a first nucleic acid, comprising: a) the first nucleic acid; i. The 5 'end linked to a detectably tagged mobile solid support comprising a region complementary to a section of the first nucleic acid immediately 3' to the selected nucleotide, A second nucleic acid terminating at the 3 'end of the test nucleotide positioned to base pair with the nucleotide, and ii. Fluorescently labeled third nucleic acid comprising a region complementary to the section of the second nucleic acid immediately 5 ′ to and adjacent to the selected nucleotide, is added to the first and second nucleic acids. Contacting the first nucleic acid with the third nucleic acid under hybridization conditions to form a hybridization product; and b) adding ligase to the hybridization product under ligation conditions. And c) detecting the presence or absence of a fluorescent label after dissociating the hybridized nucleic acid in the nucleic acid linked to the movable solid support, and detecting the presence of the label by using the labeled third nucleic acid and the movable solid support. Exhibiting linkage with a second nucleic acid linked to a phase support, and wherein the characteristics of the test nucleotide in the second nucleic acid are associated with the selected nucleotide. Characterizing complementary nucleotides and identifying the nucleotides so selected. 22. The genomic DNA, wherein the first nucleic acid has been subjected to a viscosity reduction treatment,
Oligonucleotide, 16s ribosomal RNA, PCR product, DNA fragment, RN
22. The method according to claim 21, which is an A molecule, a cDNA molecule or a cRNA molecule. 23. A method for identifying a selected nucleotide in a first nucleic acid, comprising: a) the first nucleic acid; i. A section of the first nucleic acid linked at the 5 'end to a detectably tagged mobile solid support and linked at the 3' end to a fluorescent label, just 3 'to the selected nucleotide. A second nucleic acid comprising a region complementary to and terminating at the 3 'end of a test nucleotide positioned to base pair with the selected nucleotide;
And ii. A third nucleic acid that is fluorescently labeled at the 5 ′ end, including a region complementary to the section of the second nucleic acid immediately 5 ′ to and adjacent to the selected nucleotide; Contacting under hybridization conditions such that the second nucleic acid forms a hybridization product and the first nucleic acid and the third nucleic acid form a hybridization product; and b) the 3 'end of the second nucleic acid Detecting the presence or absence of fluorescent energy transfer between the fluorescent label at the 5 ′ end of the third nucleic acid and the fluorescent label at the 5 ′ end of the third nucleic acid. Show,
Identifying characteristics of the test nucleotide hybridized in the second nucleic acid with characteristics of a nucleotide complementary to the selected nucleotide, and identifying the nucleotide selected in the manner described above. 24. A method for identifying a selected nucleotide in a first nucleic acid, comprising: a) the first nucleic acid; i. A region complementary at the 5 'end to a detectably tagged mobile solid support and complementary to a section of the first nucleic acid immediately 3' to and immediately adjacent to the selected nucleotide. A second nucleic acid comprising: and ii. Combining a fluorescently labeled third nucleic acid comprising a region complementary to the section of the second nucleic acid immediately 5 ′ to and adjacent to the selected nucleotide into a first nucleic acid and a second nucleic acid; The first, second and third nucleic acids are contacted with the selected nucleotide by forming a hybridization product and contacting the first nucleic acid and the third nucleic acid under hybridization conditions to form a hybridization product. Forming a hybridization product defining the opposite gap; b) adding test nucleotides, polymerase and ligase under polymerization and ligation conditions; and c) in the nucleic acid linked to said mobile solid support. After dissociating the hybridized nucleic acid, the presence or absence of a fluorescent label is detected, and the presence of the label indicates that the test nucleic acid and the second nucleus are present. 2 shows polymerization with an acid and ligation of the labeled third nucleic acid with a second nucleic acid linked to the mobile solid support, wherein the characteristics of the test nucleotide are complementary to the selected nucleotide. Characterizing the nucleotides and identifying the nucleotides so selected. 25. A method for quantifying the expression of a selected nucleic acid in a sample, comprising: a) (i) a message nucleic acid isolated from a selected source; and (ii) a detectable tag. Contacting a nucleic acid probe that is linked at the 5 'end to the movable mobile solid support and that includes a region complementary to a section of the selected nucleic acid; b) claim 1,13,17,19,21, Any one of 22, 23 and 24
And c) quantifying the fluorescence attached to the mobile solid support, and the amount of the fluorescence is indicative of the amount of the selected nucleic acid in the sample. 26. The method of claim 26, wherein the message nucleic acid is mRNA, cRNA or cDN.
26. The method of claim 25, wherein A is. 27. A method for detecting the result of an identification reaction to identify selected nucleotides in a target nucleic acid, comprising: a) a first complementary region and a second complementary region, wherein the second complementary region The region is 5 'to the first complementary region and the first complementary region is immediately 3' of the selected nucleotide.
(Including a region complementary to a section of the target nucleic acid on the side and adjacent thereto) and a sample containing the target nucleic acid, the first complementary region of the target oligonucleotide and the target oligonucleotide Contacting under hybridization conditions to form a hybridization product between a target nucleic acid region complementary to the first complementary region to form a first hybridization product; b) said first hybridization product Performing a selected identification reaction such that a selectively labeled detection product comprising the second complementary region of the target oligonucleotide can be formed using a DNA to characterize the selected nucleotide; c) The detection product is covalently linked to a mobile solid support and complementary to the second complementary region of the target oligonucleotide. Isolating said detection product by contacting a capture oligonucleotide comprising an acid sequence under hybridization conditions to form a second hybridization product; and d) labeling said second hybridization product Detecting and / or identifying the label of said detection product, wherein the presence and / or characteristic of the label is indicative of a characteristic of the selected nucleotide in the target nucleic acid. 28. The method according to claim 27, wherein said identification reaction is an oligonucleotide ligation reaction. 29. The identification reaction comprises performing a primer extension reaction under primer extension conditions using a detectably labeled end-of-chain nucleotide with the first hybridization product. And wherein the detecting comprises detecting the presence or absence of a label incorporated into the second hybridization product, wherein the presence of the label indicates that the labeled nucleotide is incorporated into the first hybridization product. 28. The method of claim 27, wherein the characteristics of the incorporated labeled nucleotides indicate characteristics of nucleotides complementary to the selected nucleotides and thus identify the selected nucleotides in the target nucleic acid. Method. 30. The target nucleic acid is an oligonucleotide, 16s ribosomal RNA, PCR product, DNA fragment, RNA molecule, cDNA molecule or cRNA molecule, and the nucleic acid primer is an oligonucleotide, PCR product, DNA fragment, RNA molecule. 28. The method of claim 27, which is a cDNA molecule or a cRNA molecule. 31. The method of claim 27, wherein said mobile solid support is a bead. 32. The method of claim 31, wherein said beads are polystyrene-divinylbenzene. 33. The method of claim 27, wherein said mobile solid support is detectably dyed, radiolabeled, or magnetically tagged. 34. The method according to claim 27, wherein said first nucleic acid is an amplification product. 35. The method according to claim 27, wherein said first nucleic acid is a PCR product. 36. The method of claim 27, wherein the detection is performed by passing the movable solid support over a laser detection device capable of detecting / identifying a detectable tag. 37. The detection is performed by fixing the movable solid support on a two-dimensional surface and passing a laser detection device capable of detecting / identifying a detectable tag on the solid support. 28. The method according to 27. 38. A method for detecting a reaction product to identify a selected nucleotide in a target nucleic acid, comprising: a) a first complementary region and a second complementary region, wherein the first complementary region is An oligonucleotide primer, wherein the second complementary region comprises a nucleic acid sequence complementary to the capture oligonucleotide, wherein the oligonucleotide primer is complementary to a section of the target nucleic acid immediately 3 'to and adjacent to the selected nucleotide. A target oligonucleotide containing a target nucleic acid, and a sample containing the target nucleic acid.
Contacting under a hybridization condition to form a hybridization product between a first complementary region of the target oligonucleotide and a target nucleic acid region complementary to the first complementary region of the target oligonucleotide, the first hybridization product B) performing a primer extension reaction under primer extension conditions using said first hybridization product and a detectably labeled end-of-chain nucleotide to form a primer extension product. C) combining the primer extension product with a capture oligonucleotide covalently linked to a mobile solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to a second complementary region of a target oligonucleotide. Contacting under hybridization conditions, Isolating the primer extension product by forming an isolated second hybridization product; and d) detecting the presence or absence of a label in the isolated second hybridization product, and detecting the presence of the label. Indicates that the labeled nucleotide is incorporated into the primer extension product, and wherein the characteristics of the identified incorporated labeled nucleotide indicate characteristics of a nucleotide complementary to the selected nucleotide, and Identifying a selected nucleotide in the target nucleic acid. 39. The target nucleic acid is an oligonucleotide, a 16s ribosomal RNA, a PCR product, a DNA fragment, an RNA molecule, a cDNA molecule or a cRNA molecule, and the nucleic acid primer is an oligonucleotide, a PCR product, a DNA fragment, an RNA molecule. 39. The method of claim 38, which is a cDNA or cRNA molecule. 40. The method of claim 39, wherein said labeled chain terminating nucleotide is a 3 ′ deoxynucleotide, 3 ′ deoxyribonucleotide, thiol nucleotide derivative or dideoxynucleotide. 41. The chain terminating nucleotide is a dideoxynucleotide, and the primer extension is performed in the presence of one identified dideoxynucleotide that has been labeled and three different unlabeled dideoxynucleotides. 39. The method of claim 39. 42. The chain terminating nucleotide is a dideoxynucleotide and the primer extension is labeled with a first identified dideoxynucleotide labeled with a first detectable label, a second detectable label. The second identified dideoxynucleotide, the third identified dideoxynucleotide labeled with a third detectable label, and the fourth identified dideoxynucleotide labeled with a fourth detectable label And the detection of the presence of the first, second, third or fourth label detectable in the second hybridization product is such that the nucleotide complementary to the selected nucleotide is the first nucleotide, respectively. 1, second,
40. The protein is identified as being identified as a third or fourth dideoxynucleotide.
The method described in. 43. The method of claim 39, wherein said mobile solid support is a bead. 44. The method of claim 43, wherein said beads are polystyrene-divinylbenzene. 45. The method of claim 43, wherein said mobile solid support is detectably labeled with a dye, radiolabel, or magnetic tag. 46. The method of claim 39, wherein said target nucleic acid is an amplification product. 47. The method of claim 39, wherein said target nucleic acid is a PCR product. 48. The method of claim 39, wherein the detection is performed by passing the movable solid support over a laser detection device capable of detecting / identifying a detectable label. 49. The detection is performed by fixing the movable solid support on a two-dimensional surface and passing a laser detection device capable of detecting / identifying a detectable label on the solid support. 39. The method of claim 39. 50. A method for detecting a result of an identification reaction to identify a selected nucleotide in a target nucleic acid, comprising the steps of: a) (i) a first complementary region and a second complementary region (wherein The complementary region comprises a region complementary to the section of the target nucleic acid immediately 3 'to and adjacent to the selected nucleotide, and the second complementary region comprises a nucleic acid sequence complementary to the capture oligonucleotide). And (ii) a fluorescently labeled reporter oligonucleotide comprising a region complementary to a section of the target nucleic acid immediately 5 ′ to and adjacent to the selected nucleotide. And a target nucleic acid region complementary to the first complementary region of the target oligonucleotide. Under hybridization conditions that specifically hybridize between the sections of the target nucleic acid that are complementary to the lignonucleotide and the reporter oligonucleotide, hybridize with the sample containing the target nucleic acid to eliminate opposing gaps of the selected nucleotide. Forming a defined first hybridization product; b) adding the identified test nucleotides, polymerase and ligase under polymerization and ligation conditions to form a labeled product; c) forming the hybridized nucleic acid. Dissociating; d) the labeled product and a capture oligonucleotide covalently linked to a mobile solid support, wherein the capture oligonucleotide is a nucleic acid sequence complementary to a second complementary region of the target oligonucleotide. And hybridizing Isolating said labeled product by contacting under hybridization conditions to form a second hybridization product; and e) detecting the presence or absence of the label in said second hybridization product, Indicates the polymerization of the identified test nucleotide and the target oligonucleotide, and the ligation of the labeled reporter oligonucleotide and the polymerized target oligonucleotide, wherein the characteristics of the identified test nucleotide are selected. Characterizing nucleotides complementary to the selected nucleotides and thus identifying the selected nucleotides in the target nucleic acid. 51. The target nucleic acid is an oligonucleotide, a 16s ribosomal RNA, a PCR product, a DNA fragment, an RNA molecule, a cDNA molecule or a cRNA molecule, and the nucleic acid primer is an oligonucleotide, a PCR product, a DNA fragment, an RNA molecule. 51. The method of claim 50, which is a cDNA molecule or a cRNA molecule. 52. The reporter oligonucleotide is selected from the group consisting of 1024 sets of all possible pentameric oligonucleotides.
A method according to claim 50. 53. The method of claim 50, wherein said mobile solid support is a bead. 54. The method of claim 53, wherein said beads are polystyrene-divinylbenzene. 55. The method of claim 50, wherein said mobile solid support is detectably labeled with a dye, radioactive label, or magnetic tag. 56. The method of claim 50, wherein said target nucleic acid is an amplification product. 57. The method of claim 50, wherein said target nucleic acid is a PCR product. 58. The method of claim 50, wherein the detection is performed by passing the movable solid support over a laser detection device capable of detecting / identifying a detectable label. 59. The detection is carried out by fixing the movable solid support on a two-dimensional surface and passing a laser detection device capable of detecting / identifying a detectable label on the solid support. 50. The method of claim 50. 60. A method for examining polymorphism of a selected nucleotide in a genomic DNA subjected to a viscosity reducing treatment, comprising: a) a section complementary to a section of one nucleic acid strand on the 5 ′ side of the selected nucleotide. Using a first nucleic acid primer comprising a region and a second nucleic acid primer complementary to a section of the other nucleic acid strand downstream of the selected nucleotide, the nucleotides selected between the two primers Amplifying genomic DNA under conditions that specifically amplify the region to form a PCR product; b) a first complementary region and a second complementary region (wherein
Is the PCR region immediately 5 'to and adjacent to the selected nucleotide.
Contacting under hybridization conditions to form a first hybridization product; c) with said first hybridization product. Performing a primer extension reaction under primer extension conditions using a detectably labeled end-of-chain nucleotide to form a primer extension product; d) combining the primer extension product with a mobile solid support. A capture oligonucleotide, which is covalently linked to a body, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to a second complementary region of the target oligonucleotide, is isolated by contact under hybridization conditions. By forming a second hybridization product Isolating the primer extension product; e) detecting the presence or absence of a label incorporated into the second hybridization product, and detecting the presence of the label, such that the labeled chain terminating nucleotide is incorporated into the primer extension product. And wherein the characteristics of the incorporated labeled end-of-chain nucleotides are characteristic of nucleotides complementary to the selected nucleotides; and f) the characteristics of the selected nucleotides are non-polymorphic nucleotides. And wherein the characteristics of the selected nucleotide are different from those of the non-polymorphic nucleotide as compared to indicating a polymorphism of the selected nucleotide. 61. The method of claim 60, wherein said labeled chain terminating nucleotide is a 3 'deoxynucleotide, 3' deoxyribonucleotide, thiol nucleotide derivative or dideoxy nucleotide. 62. The method of claim 60, wherein the DNA has been sheared to reduce viscosity. 63. The method of claim 60, wherein the DNA has been treated with DNase to reduce viscosity. 64. A method for examining polymorphisms of selected nucleotides in genomic DNA that has been subjected to a viscosity reduction treatment, comprising: a) a first region having a T7 RNA polymerase promoter, and 5 minutes immediately adjacent to the selected nucleotides. An oligonucleotide containing a second region complementary to a section of one nucleic acid strand on the 'side is used as a primer, and one of the strands is cRN
A using T7 RNA polymerase to amplify to A, and using a reverse transcriptase to amplify the other strand complementary to the cRNA strand, under the conditions that specifically amplify the nucleotide region between the two primers, Amplifying the DNA to form an amplification product; b) said amplification product and a first and a second complementary region, wherein the first complementary region is immediately 5 ′ to the selected nucleotide. And a second complementary region that is complementary to a section of one strand of the PCR product adjacent thereto and the second complementary region is 5 ′ to the first complementary region). Forming a first hybridization product with the first hybridization product; c) identifying the first hybridization product with a detectably labeled chain termination nucleus. Performing a primer extension reaction under primer extension conditions using an otide to form a primer extension product; d) a capture oligonucleotide covalently bound to a movable solid support with the primer extension product (here, capture The oligonucleotide comprising a nucleic acid sequence complementary to the second complementary region of the target oligonucleotide) under hybridization conditions to form an isolated second hybridization product. Isolating the product; e) detecting the presence or absence of a label incorporated in the second hybridization product and detecting the presence of the label that the labeled chain terminating nucleotide is incorporated in the primer extension product. Indicates the characteristics of the incorporated labeled chain terminating nucleotide. Exhibits the characteristics of the nucleotide that is complementary to the selected nucleotide; and f) comparing the characteristics of the selected nucleotide to the non-polymorphic nucleotide and determining that the characteristics of the selected nucleotide are those of the non-polymorphic nucleotide. If the characteristics are different, a polymorphism of the selected nucleotide is indicated. 65. The method of claim 64, wherein said labeled chain terminating nucleotide is a 3 ′ deoxynucleotide, 3 ′ deoxyribonucleotide, thiol nucleotide derivative or dideoxynucleotide. 66. A method for examining polymorphism of selected nucleotides in a genomic DNA subjected to a viscosity reduction treatment, comprising: a) genomic DNA, a first complementary region and a second complementary region, wherein The first complementary region is complementary to a section of one strand of the PCR product immediately 5 ′ to and adjacent to the selected nucleotide, and the second complementary region is 5 ′ to the first complementary region. Contacting under hybridization conditions to form a specific first hybridization product; b) detecting the specific first hybridization product with a detectable first hybridization product. Performing a primer extension reaction under primer extension conditions using the identified chain terminating nucleotide labeled as above; c) Contacting a capture oligonucleotide covalently attached to a solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to the second complementary region of the target oligonucleotide, under hybridization conditions Isolating the primer extension product by forming an isolated second hybridization product; d) detecting the presence or absence of a label incorporated in the second hybridization product, Indicating that the labeled chain terminating nucleotide is incorporated into the hybridization product, wherein the characteristic of the incorporated labeled chain terminating nucleotide is characteristic of a nucleotide complementary to the selected nucleotide; And e) determining the characteristics of the selected nucleotides from the non-polymorphic nucleic acid. Comparing the selected nucleotide with a non-polymorphic nucleotide as compared to an otide, indicating a polymorphism of the selected nucleotide. 67. The method of claim 66, wherein said labeled chain terminating nucleotide is a 3 ′ deoxynucleotide, 3 ′ deoxyribonucleotide, thiol nucleotide derivative or dideoxynucleotide. 68. A method for identifying selected nucleotides in a target nucleic acid, comprising: a) a target nucleic acid; i. A first complementary region and a second complementary region, wherein the first complementary region is complementary to a section of the first nucleic acid immediately 5 ′ to the selected nucleotide and the target oligonucleotide is selected A target oligonucleotide terminated at the 3 'end of the identified test nucleotide positioned to base pair with the second nucleotide and the second complementary region is 5' to the first complementary region). And ii. A fluorescently labeled reporter oligonucleotide comprising a region complementary to the section of the target nucleic acid immediately 3 ′ to and adjacent to the selected nucleotide, hybridizing the target nucleic acid with the target oligonucleotide; and Contacting a target nucleic acid with a reporter oligonucleotide under hybridization conditions to form a first hybridization product; b) adding a ligase to said first hybridization product under ligation conditions; c. 2.) The first hybridization product and a capture oligonucleotide covalently linked to a mobile solid support, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to a second complementary region of the target oligonucleotide. And the hybridization Isolating said first hybridization product by contacting under contact conditions to form an isolated second hybridization product; and d) hybridizing in said second hybridization product. Detecting the presence or absence of a fluorescent label after dissociating the nucleic acid, the presence of the label indicates the link between the labeled reporter oligonucleotide and the target oligonucleotide, and the characteristics of the test nucleotide in the target oligonucleotide are selected. Characterizing nucleotides complementary to the selected nucleotides and identifying the nucleotides so selected; 69. The genomic DNA, wherein the first nucleic acid has been subjected to a viscosity reduction treatment,
Oligonucleotide, 16s ribosomal RNA, PCR product, DNA fragment, RN
69. The method according to claim 68, which is an A molecule, a cDNA molecule or a cRNA molecule. 70. A method for identifying selected nucleotides in a target nucleic acid, comprising: a) a target nucleic acid; i. Linked to the fluorescent label at the 3 'end, comprising a first complementarity region complementary to the section of the target nucleic acid immediately 3' to the selected nucleotide, and arranged to base pair with the selected nucleotide. A target oligonucleotide terminating at the 3 'end of a test nucleotide and having a second complementary region 5' to the first complementary region;
And ii. A reporter oligonucleotide, which is 5'-end and includes a region complementary to a section of the target nucleic acid immediately adjacent to the selected nucleotide and fluorescently labeled at the 5 'end, hybridizes the target nucleic acid and the target oligonucleotide. And contacting the target nucleic acid with a reporter oligonucleotide under hybridization conditions to form a first hybridization product; b) attaching the first hybridization product to a mobile solid support; A covalently attached capture oligonucleotide, wherein the capture oligonucleotide comprises a nucleic acid sequence complementary to the second complementary region of the target oligonucleotide, is contacted under hybridization conditions with the isolated oligonucleotide. Form 2 hybridization products D) isolating the first hybridization product by a fluorescent label at the 3 ′ end of the target oligonucleotide and a 5 ′ end of the reporter oligonucleotide in the second hybridization product. Detecting the presence or absence of a fluorescent energy transfer between a certain fluorescent label and the presence of the fluorescent energy transfer indicates hybridization between the identified test nucleotide and the target nucleic acid, and for the hybridized test nucleotide in the target oligonucleotide. Identifying the nucleotides that are complementary to the selected nucleotides and identifying the nucleotides thus selected. 71. A method for identifying selected nucleotides in a target nucleic acid, comprising: a) a target nucleic acid; i. A first complementarity region and a second complementarity region 5 'to the first complementarity region, wherein the first complementarity region is a region of the target nucleic acid immediately 3' to and directly adjacent to the selected nucleotide. A target oligonucleotide, which is complementary to the section)
And ii. A fluorescently labeled reporter oligonucleotide comprising a region complementary to the section of the second nucleic acid immediately 5 ′ to and adjacent to the selected nucleotide is used to bind the target nucleic acid and the hybridization product to the target oligonucleotide. Forming and contacting the target nucleic acid and the reporter oligonucleotide under hybridization conditions to form a hybridization product, wherein the target nucleic acid, the target oligonucleotide and the reporter oligonucleotide define an opposing gap of the selected nucleotide. Forming hybridization products; b) adding the identified test nucleotides, polymerase and ligase under polymerization and ligation conditions; c) sharing said first hybridization product with a mobile solid support. A second capture oligonucleotide isolated by contacting with a bound capture oligonucleotide (where the capture oligonucleotide comprises a nucleic acid sequence complementary to the second complementary region of the target oligonucleotide) under hybridization conditions Isolating the first hybridization product by forming a hybridization product of the above; and d) detecting the presence or absence of a fluorescent label in the second hybridization product after dissociating the hybridized nucleic acid Wherein the presence of a label indicates the polymerization of the test nucleic acid with the target oligonucleotide, and the ligation of the labeled reporter oligonucleotide with the target oligonucleotide linked to the mobile solid support, Nucleotide characteristics change the selected nucleotide Characterizing the complementary nucleotides and identifying the nucleotides so selected. 72. A method for quantifying the expression of a selected nucleic acid in a sample, comprising: a) (i) a message nucleic acid isolated from a selected source; (ii) a first complementary region and a first nucleic acid. Contacting with a target oligonucleotide comprising a second complementary region 5 'to the complementary region of (where the first complementary region comprises a region complementary to a section of the selected nucleic acid); b) The first hybridization product is used to perform a selected identification reaction that includes the second complementary region of the target oligonucleotide and that can form a selectively labeled detection product, and C) a capture oligonucleotide covalently linked to said detection product and a mobile solid support and comprising a nucleic acid sequence complementary to a second complementary region of a target oligonucleotide Isolating said detection product by contacting under hybridization conditions to form an isolated hybridization product; and d) quantifying the fluorescence in said isolated hybridization product, The amount of fluorescence indicating the amount of the selected nucleic acid in the sample. 73. The message nucleic acid is mRNA, cRNA or cDN.
73. The method of claim 72, wherein A.