JP2002508664A - Method for detecting multiple single nucleotide polymorphisms in a single reaction - Google Patents

Abstract

  (57) [Summary] Molecules and methods suitable for identifying multiple polymorphic sites in the genome of a plant or animal. Identification of such sites is useful for determining identity, ancestry, predisposition to a genetic disease, the presence or absence of a desired attribute, and the like.

Description

DETAILED DESCRIPTION OF THE INVENTIONTitle of invention Method for detecting multiple single nucleotide polymorphisms in a single reaction TECHNICAL FIELD OF THE INVENTION   The present invention is in the field of recombinant DNA technology. For more information, see the book The invention relates to one or more single nucleotides in the genome of a plant, animal or microorganism. Identify leotide polymorphisms in a single reaction and identify such sites with identity, ancestry or genetic Molecules and methods suitable for use in analyzing traits.Cross-reference of related applications   This application claims priority with US Ser. No. 08 / 216,538, filed Mar. 23, 1994. (The application is filed in U.S. patent application Ser. No. 08 / 145,145 (November 3, 1993) Application) is a partial continuation application).Background of the Invention   Being able to determine the genotype of an animal, plant or microorganism is forensic, medical and In epidemiology and public health, and in animal breeding and exhibition It is basically important. Such determinants include, for example, infection To identify the etiology of a sexual illness, to determine if two individuals are related, Or it is necessary to map a gene into the genome of an organism.   Analyzes of identity and parenthood along with being able to diagnose the disease are also human , Animal and plant genetic studies, especially legal or paternity Central interest in assessment and in assessing an individual's risk of developing a genetic disease It is also a thing. Such a goal is to make the DNA of one individual different from the DNA of another individual. It has been pursued by analyzing the DNA sequence for mutations that have been deemed to have been.   If such a mutation results in a fragment produced by restriction endonuclease cleavage If it changes length, such a mutation is called a restriction fragment length polymorphism ("RFLP"). You. RFLP is widely used in human and animal genetic analysis (Grassberg , J., UK Patent Application No. 2135774; Skolnick, M.H. et al.Cytogen . Cell Ge net .32: 58-67 (1982); Bostein, D. et al.Ann . J. Hum. Genet.32: 314-331 (19 80); Fischer, S .; G. et al. (PCT application WO 90/13668); Uhlen, M., PCT application WO 90/11369). Linking heritable attributes to specific RFLPs If linked, the presence of the RFLP in the target animal indicates that the animal Can be used to predict that it is likely to show Multiple a RFLP multiple loci to map complex traits dependent on rel (Multilocus) Statistical methods have been developed that allow analysis (Lander, S. et al.P roc . Natl. Acad. Sci. (USA) 83: 7353-7357 (1986); Lander, S. et al.Proc . N atl. Acad. Sci. (USA) 84: 2363-2367 (1987); Donis-Keller, H. et al.Cell 51: 3 19-337 (1987); Lander, S. et al.Genetics 121: 185-199 (1989), all referenced Cited herein). Such methods are commonly used to develop genetic maps. And can be used to develop plants or animals with more desirable attributes. Kirun (Donis-Keller, H. et al.Cell 51: 319-337 (1987); Lander, S. et al.Genetics 121 : 185-199 (1989)).   In some cases, mutations in the DNA sequence are caused by tandem dinucleotides of nucleotides. Tandem repeats containing short or trinucleotide repeat motifs (shortt andem repeats (“STR”). These tandem lipi The note also states "variable number tandem repeat ( Also referred to as "VNTR"). VNTR is used for identification and paternity analysis (Weber, J.L., U.S. Pat. No. 5,075,217; Armour, J.A.L., et al.)FEBS Lett .307: 113-115 (1992); Jones, L. et al.Eur . J. Haematol.39: 144〜14 7 (1987); Horn, G.T., et al., PCT Application WO 91/14003; Jeffreys, A.J. Jeffreys, A.J., U.S. Patent No. 5,175. No. 082; Jeffreys, A.J. et al.Amer . J. Hum. Genet.39: 11-24 (1986); Jeffr eys, A.J. et al.Nature 316: 76-79 (1985); Gray, I.C., et al.Proc . R. Acad. Soc. Lond .243: 241-253 (1991); Moore, S.S., et al.Genomics Ten: 654-660 (1991); Jef freys, A.J. et al.Anim . Genet.18: 1-15 (1987); Hillel, J. et al.Anim . Genet.Two 0 : 145-155 (1989); Hillel, J. et al.Genet.124: 783-789 (1990)), currently many It has been used in many gene mapping studies.   A third class of DNA sequence variation is the occurrence of single nucleotides present between individuals of the same species. De-polymorphism ("SNP"). Such polymorphisms are identified by STR or V It occurs much more frequently than NTR. In some cases, such polymorphisms Contain mutations that are a critical feature in genetic diseases. In fact, such a change The difference is sufficient to actually cause the disease (ie, hemophilia, sickle cell anemia, etc.). Affects a single nucleotide in a protein-encoding gene in a sensitive way. Many In many cases, these SNPs occur in non-coding regions of the genome.   Despite the central importance of such polymorphisms in modern genetics, Enable analysis of one or more of these loci in a single reaction format No practical method has been developed.   The present invention provides such an improved method. In fact, the present invention Genes identified and paternally identified by discriminating single nucleotide polymorphism variants Methods and gene sequences are provided that enable analysis and diagnosis of disease.Summary of the Invention   The present invention includes single nucleotide polymorphisms (SNPs) present in all life forms. About molecules. The invention provides (i) one or more novel single nucleotides A method for identifying polymorphisms, (ii) repeated analysis and testing of these SNPs in different samples And (iii) determining the presence of such sites in animals, plants and microorganisms. How to use for child analysis.   Analysis of such sites (genotyping) can include identity, ancestry, predisposition to genetic disease, Useful for determining the presence or absence of a desired attribute, and the like. Specifically, the present invention One or more nucleotide sequences of the genomic DNA segment of any organism One or more interrogations having complementary polynucleotide sequences (Interrogation) providing nucleic acid (or nucleic acid analog) primer molecules Wherein the genomic segment is a mammalian single nucleotide polymorphism array. Located just 3 ′ distal to the single nucleotide polymorphism site X of the Or nucleotide analog) for nucleic acid (or nucleic acid analog) primer The template-dependent extension of the primer allows the primer molecule to bind to a single nucleotide (or analog). And the single nucleotide (or analog) is replaced with the single nucleo Complementary to nucleotide X of the Tide polymorphism allele.   The present invention provides for template-dependent extension of primers using ddATP, ddTTP, dd. Selected from the group consisting of CTP and ddGTP (or other chain terminating base analog) One or more dideoxynucleotide triphosphate derivatives (or analogs) G) but in the absence of dATP, dTTP, dCTP and dGTP It relates to an aspect.   The present invention further provides for one or more single nucleotide polymorphisms in a single reaction. A method of identifying (A) one or more identifiable interrogation oligonucleotides ( Or oligonucleotide analog) primers to one or more target nucleic acids Hybridize to the molecule, where each oligonucleotide primer is The 3 'end of the primer is immediately adjacent to the specific and unique target nucleotide of interest Complementary to a specific and unique region of each target nucleic acid molecule, such as (B) Each interrogation oligonucleotide (or analog) is template dependent With one or more non-extendable polymerases Occurs in the presence of a nucleotide (or nucleotide analog) of (C) For each interrogation primer used, such a primer Of non-extendable nucleotides (or nucleotide analogs) introduced into Each nucleotide (or analog) of interest by determining the identity And identifying the non-extendable nucleotide (or nucleotide Nalog) is complementary to the target nucleotide of the primer; (D) on a suitable matrix or other standard method of physical or chemical separation or Separates the extended primer by an identification method including.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows a preferred method for cloning random genomic fragments. Genomic DNA is subjected to size fractionation and then plasmids are used to obtain random clones. Introduce into the vector. PCR sequencing to determine the sequence of the inserted genomic sequence Design and use limers.   Figure 2 shows the cycle sequencing of random genome fragments. ) Obtained by a preferred method for identifying a novel polymorphic sequence. Shows the data obtained.   FIG. 3. RFL for screening random clones for polymorphic sequences. The P method is shown.   FIG. 4 shows that for a given panel of genetic markers, two individuals have the same genotype 3 shows a graph of the probability that the probability will be high.   FIG. 5 shows that a given panel of 20 genetic markers is not a problem for mothers Probability of rejecting random and suspicious fathers in paternity lawsuits The graph is shown.   FIG. 6 shows a preferred method for genotyping SNPs. The seven steps are: Figure 4 shows how to perform GBA starting from a biological sample.Description of the preferred embodiment I. Advantages of the Single Nucleotide Polymorphism of the Invention and Its Use in Gene Analysis   A. Polymorphic attributes   Certain gene sequences of interest to the present invention are single nucleotide polymorphisms. "including. A “polymorphism” is a variation in the DNA sequence of some member of a species. You. Animal and plant genomes have undergone natural mutations during their sustained evolution. (Gusella, J.F.,Ann . Rev. Biochem.55: 831-854 (1986)). Such a spike Most mutations, however, produce polymorphisms. The mutated sequence and the original sequence are Co-existing in a group. In some cases, such coexistence is stable or near It is in stable equilibrium. In other cases, such mutations are not Will eventually provide an advantage to evolution (ie, ), It will be integrated into the genome of all members of that species.   Therefore, a polymorphism is that some members of certain species are not mutated due to the existence of the polymorphism. Other members have a mutated sequence (ie, the original "allele") That is, "allelic" in that it has a mutated "allele"). ". In the simplest case, there is only one mutated sequence and this multiple The type is called dialelic. When another mutation occurs, (Triallelic), etc. The allele is defined by the nucleotide containing the mutation. expressed.   The present invention relates to specific classes of allelic variants and to the inheritance of plants, animals, or microorganisms. It relates to its use for determining gene types. Such allelic variants are described herein. Are referred to as "single nucleotide polymorphisms" or "SNPs." "Single nucleo The "tide polymorphism" is defined by the following attributes. The central attribute of such polymorphisms is It is to include a polymorphic site “X” which is a site of mutation between allele sequences. SNP's The second property is that the polymorphic site "X" often precedes the "unmutated" sequence of the allele. It has been followed. Therefore, the polymorphic site of the SNP is a "5 'proximal" non- Located “immediately” 3 ′ to the mutated sequence and “immediately” 5 ′ to the “3 ′ distal” unmutated sequence It is assumed to be located. Such a sequence flanks the polymorphic site. Single The term "single" of a nucleotide polymorphism refers to the number of nucleotides in the polymorphism (ie, one Nucleotides); this is the number of polymorphisms (from one to many) present in the target DNA. Has no relation to numbers).   As used herein, an allele is referred to as an "allele" if the sequence does not change within that species population. The sequence is referred to as the 'unmutated' sequence, and if mapped, the Maps to the "corresponding" sequence of the same allele in the nom. Two or more S Note that the NPs may be located very close to each other. Two arrays Are `` corresponding '' when they are analogs from different sources. It is called a row. The gene sequence encoding hemoglobin in two humans 9 is an example of an allele sequence. The definition of “corresponding allele” in this specification is To change the meaning of a word that is understood by those skilled in the art, to clarify the meaning of the word It is not intended. Each column in Table 1 shows the “corresponding” equine allele polymorphism site. Nucleotide identification and unmutated 5 'proximal and 3' distal sequences (also in the S NP). "Corresponding alleles" for human alleles are shown in Table 2. is there. Each column in Table 2 shows the identification sequence of the nucleotide at the polymorphic site of the “corresponding” human allele. Shows the unmutated 5 'proximal and 3' distal sequences, which are also attributes of the SNP. You.   Since genomic DNA is double-stranded, each SNP has a positive or negative strand. Either can be defined. Therefore, for each SNP, one strand is immediately 5 ′ The other strand will contain the immediate 3 'distal non-mutated sequence. each In a preferred embodiment where the SNP polymorphism site "X" is a single nucleotide, Each strand of P double-stranded DNA has an immediate 5 ′ proximal unmutated sequence and an immediate 3 ′ distal unmutated Will contain both sequences.   Preferred SNPs of the present invention are other nucleotides of one nucleotide at the SNP polymorphism. Regarding substitution with tides, SNPs are also more complex and Deletion of nucleotides from one of the two corresponding sequences or two corresponding sequences May include an insertion into one of the For example, in certain animals Rows contain A in a particular polymorphic site, whereas in other animals a single Or there may be multiple base substitutions. Preferred SNPs of the invention are non-mutated proximal SNPs have both non-mutated proximal sequences or both It may have only the non-mutated distal sequence.   Nucleic acid molecules having a sequence complementary to the immediate non-mutated sequence immediately 3 ′ to the SNP are identified as When extended in a "type-dependent" manner, the extension product containing the polymorphic site of the SNP Can be formed. A preferred example of such a nucleic acid molecule is the 5 'proximal non-mutated A nucleic acid molecule having the same sequence as the sequence. "Template-dependent" extension refers to The polymerase extends the primer so that the sequence is complementary to the sequence of the nucleic acid template. Means that it can mediate. A “primer” is a nucleic acid in a “template-dependent” extension reaction. Can be extended by the covalent attachment of a nucleotide (or nucleotide analog) , Single-stranded oligonucleotide (or oligonucleotide analog) or single-stranded Refers to a strand polynucleotide (or polynucleotide analog). Such noh To be powerful, the primer must be 3 'hydroxyl (or polymerase mediated It must have a terminus for the other nucleic acid molecule (another chemical group suitable for extension). That is, it must be hybridized to a "template"). The primer is (1) The 3 'end of the primer is immediately adjacent to the target nucleotide of interest. As such, a unique 8 base or longer length complementary to a particular region of the target molecule Sequence and (2) specific and unique length, physical or chemical properties, neutral components 5 'tail. Most preferably, the complementary region of the primer is about 2 It has zero bases, but may be a shorter or longer primer. Typically, The complementary region of the primer is from about 12 to about 20 bases. 5 'tail neutral The components are non-specific and non-hybridizing, such as poly T, non-basic residues, etc. A polymer or a chemical group. "Polymerase" means that a nucleic acid molecule When hybridized to the template nucleic acid molecule, the nucleoside triphosphate (or To extend the 3 'hydroxyl group of the nucleic acid molecule by introducing Is an enzyme. The polymerase enzyme is available from Watson, J.D.,In: Molecular Biology o f the Gene Third Edition, Benjamin, Menlo Park, California (197 7) (discussed herein by reference) and similar sources I have. Commonly known as other polymerases, for example, "Klenow" polymerase Large Proteolytic Fragment of DNA Polymerase I from Escherichia coli DNA polymerase I, bacteriophage T7 DNA polymerase, etc. Can also be used to perform the methods described herein. Immediately after SNP A nucleic acid having the same sequence as the 3 ′ distal non-mutated sequence has the same sequence as the immediately 5 ′ proximal sequence. Template-dependent primer with one nucleotide extended in a template-dependent manner Can be ligated in any way.   B. Benefits of using SNPs for gene analysis   The single nucleotide polymorphism site of the present invention can be used for plant, animal, or microbial DNA. Can be used for analysis. Such sites include mammals, including humans, Mammals, livestock (dogs, cats, etc.), farm animals (cows, sheep, etc.) and other Suitable for analyzing genomes of animals of economic importance. However, the present invention Single nucleotide polymorphism sites are useful for other types of animals, plants, and microorganisms Can be. How many SNPs are in gene analysis compared to STR and VNTR? It has that distinct advantage.   First, SNPs are more frequent (about 10 to 100) than STRs and VNTRs. Times higher) and more uniformly. The higher frequency of SNPs indicates that SNPs It means that it can be more easily identified than the class polymorphism. The distribution is more even One is to identify SNPs "closer" to a particular attribute of interest Enable. The combination of these two attributes makes SNPs extremely valuable I do. For example, certain genes with certain attributes (for example, predisposition to cancer) If it reflects a mutation at the locus, use all polymorphisms associated with the locus One can predict the probability that an individual will exhibit the trait.   The value of such predictions is determined in part by the rejection between the polymorphism and the locus. Is done. Therefore, if the locus is any repetitive tandem nucleotide sequence VNTR analysis has only limited value if it is far from the motif Would be. Similarly, if the locus moves away from any detectable RFLP If so, the RFLP analysis will not be accurate. However, The light SNP is present at about every 300 bases in the mammalian genome, and has a uniform distribution. SNPs are statistically considered to be 150 salts of a particular genetic disorder or mutation. Can be found in the group. In fact, a particular mutation may itself be a SNP. So Therefore, if the sequence of such a locus has been determined, Mutations in the nucleotides determine the quality in question.   Second, SNPs are more stable than other classes of polymorphisms. That natural sudden change The odds are about 10^-9And about 1,000 times less frequent than VNTR. Indeed, V The NTR polymorphism is characterized by a high mutation rate.   Third, SNPs further reduce their allele frequency for relatively few representative examples. Has the advantage that it can be inferred from the study of The attributes of these SNPs are RFLP Identity, paternity elimination compared to that possible with either or VNTR polymorphisms (Patternity exclusion) and analysis of animal predisposition to certain genetic attributes Enables much more advanced genetic analysis.   Fourth, SNPs are the best source of genetic information (nucleotide position and base identification). It reflects a highly efficient definition. Give such a high description Nevertheless, SNPs are much easier than RFLP or VNTR, and Detection can be performed with higher flexibility. In fact, since DNA is double-stranded, The complement of the allele can be analyzed to confirm the presence and identity of the SNP.   Flexibility in characterizing identified SNPs is a salient feature of SNPs. And For example, VNTR-type polymorphisms can be used for size-fractionation methods that can discriminate between many repetitive mutations. It can be detected more easily. RFLP is obtained by restriction digestion following size fractionation. Most easily detectable.   In contrast, SNPs can be characterized using any of a variety of methods. Such methods include direct or indirect sequencing of the site, Use of restriction enzymes when alleles create or destroy restriction sites, allele specific Use of different hybridization probes, encoded by different alleles of polymorphism Use of antibodies specific to the protein to be used, or other biochemical interpretations.   (WO 92/15712, incorporated herein by reference). "Genetic Bit Analysis" (discussed below) Analysis) ”(“ GBA ”) method is based on the nucleotides present at single nucleotide polymorphism sites. This is a method for identifying otide. GBA is the site of mutation in the target DNA sequence. The variable nucleotide in the target DNA using the nucleotide sequence information around the Oligonucleotides complementary to the region immediately adjacent to but not containing the nucleotide This is a method for elucidating polymorphic sites by designing imers. Biological target DNA template Select from the sample and hybridize to the interrogation primer. this The primer can be used to bind one or more chain-terminating nucleoside triphosphate precursors (or Single labeled dideoxy using a DNA polymerase in the presence of the appropriate analog) Extend by nucleotides (or analogs).   Cohen, D. et al. (PCT application WO 91/02087) provide for genotyping. Describes a related method by which to sequence at a desired locus. A single nucleotide with a single nucleotide using dideoxynucleotides Extend the immer. Dale et al. (PCT application WO 90/09455) disclose primers Used in conjunction with a single dideoxynucleotide species to sequence "variable sites" A method for doing so is disclosed. The method of Dale et al. Furthermore uses multiple primers. And the use of a separating element. Ritterband, M.R. (PCT application WO No. 95/17676) separates, concentrates and concentrates such target molecules in a liquid sample. An apparatus for detecting is described. Cheeseman, P. (US Pat. No. 5,302,5) No. 09) describes a related method for determining the sequence of single-stranded DNA molecules. Cheeseman's method uses a fluorescently labeled 3 'protected nucleotide triphosphate (each base is different). What With a fluorescent label).   Wallace et al. (PCT application WO89 / 10414) describe allele-specific primers. Can be used to replicate multiple regions of the target simultaneously. Several PCR methods are described. By using allele-specific primers Amplification occurs only when a particular allele is present in the sample.   Some primer-guided nucleotides for assaying polymorphic sites in DNA Introduced methods are described (Komher, J.S. et al.Nucl . Acids Res. 17: 7779-77 84 (1989); Sokolov, B.P.,Nucl . Acids Res. 18: 3671 (1990); Syvanen, A.-C. Et al.,Genomics 8: 684-692 (1990); Kuppuswamy, M.N., et al.Proc . Natl. Acad. Sci . (USA) 88: 1143-1147 (1991); Prezant, T.R., et al.Hum . Mutat. 1: 159〜164 (199 2); Ugozzoli, L. et al.GATA 9: 107-112 (1992); Nyren, P. et al.Anal . Biochem. 20 8 : 171-175 (1993)). All of these methods are used to identify bases at polymorphic sites. It differs from GBA in that it relies on the introduction of labeled deoxynucleotides. In such a format, the signal is the number of deoxynucleotides introduced. Polymorphism in runs of the same nucleotide is proportional to the length of the run. Can result in a proportional signal (Syvanen, A.-C. et al.Amer . J. Hum. Ge net. 52: 46-59 (1993)). Such a predetermined range of locus-specific signals is In the case of heterozygotes, for example, a simple three-component (2: 0, Interpretation can be more difficult compared to signals of the 1: 1 or 0: 2) class. Sa Furthermore, at certain loci, in the presence of the correct dideoxynucleotide, Even the introduction of incorrect deoxynucleotides can occur (Komher, J.S. et al.Nucl . Acids Res. 17: 7779-7784 (1989)). Such deoxynucleotide errors The introduction may, in some sequence contexts, be carried out on the DNA port to the mispaired deoxy-substrate. The Km of the lymerase is relatively small for the correctly base-paired dideoxy-substrate. m (Kornberg, A. et al., In: DNA Replication, 2nd ed. (1992) , Freeman and Company, New York; Tabor, S. et al.Proc . Natl . Acad. Sci. (USA) 86: 4076-4080 (1989)). This effect is useful for elucidating polymorphic sites. Contribute to background noise.   In contrast to all such methods, the method of the present invention involves multiple SNPs. Or very easy to determine the nucleotides. II. How to discover new polymorphic sites   A preferred method for finding polymorphic sites is to use genomes from multiple haploid genomes. Includes comparative sequencing of DNA fragments. In a preferred embodiment, as shown in FIG. In addition, such sequencing can result in 0.5 to 3 Kb of DNA from one member of a species. This is performed by preparing a random genomic library containing fragments. By the way, Using the sequences of these recombinants, the same genotype of a large number of randomly selected individuals of the species Facilitates PCR sequencing at the gene locus.   Hundreds of such genomic libraries (typically about 50,000 clones) Purify (200-500) individual clones and sequence the ends of the insert I do. To enable PCR amplification of the cloned region, only a small amount of It suffices if column information (100 to 200 bases) can be obtained. The purpose of this sequencing is to Suitable for mediating the amplification of equivalent fragments from genomic DNA samples of other members The purpose is to obtain sufficient sequence information to enable the synthesis of the primer. Preferably Such sequencing is performed using the cycle sequencing method.   D from a panel of randomly selected members of the target species using these primers Amplify NA. The number of members in the panel determines the lowest frequency of polymorphism to be isolated. You. Therefore, if 6 members are evaluated, for example, a frequency of 0.01 The existing polymorphism may not be identified. Illustrative but oversimplified mathematical treatment In the sampling of 6 members, about 0.08 (ie, a total frequency Only polymorphisms occurring more frequently than 6 members divided by 2 alleles per genome) were identified. It is expected that Therefore, if one wants to identify lower frequency polymorphisms, A number of panel members must be evaluated.   Cycle sequence analysis (Mullis, K. et al.Cold Spring Harbor Symp . Quant. Biol. 51 : 263-273 (1986); Erlich H. et al., European Patent Application No. 50,424; Europe Patent Application No. 84,796, European Patent Application No. 258,017, European Patent Application No. 2 37,362; Mullis, K., European Patent Application No. 201,184; Mullis, K. et al., United States. U.S. Patent No. 4,683,202; Erlich, H .; U.S. Patent No. 4,582,788; Saiki, R. et al., US Patent No. 4,683,194) discloses an automatic DNA sequencing apparatus and software. (Applied Biosystems, Inc.). Like that By examining the relevant parts of the chromatogram on the computer screen. Thus, differences between the sequences of different animals can be identified and confirmed. The difference is that both chains Data are available and are present in more than one haploid case in the animal population tested Only when the expression is performed, it is interpreted as reflecting the DNA polymorphism. FIG. Cycle sequencing of nome fragments to identify novel polymorphic sequences Here is a preferred method. Electroelution of PCR fragments from animals from acrylamide gel And the presence of a mixture of dNTPs and fluorescently or chemically labeled ddNTPs Sequencing using repeated cycles of thermostable Taq DNA polymerase under . The product was then separated and analyzed by Applied Biosystems, Inc. automated DNA sequencing. Analyze using a singing device. The obtained data was analyzed using ABI software. Analyze. Differences between the sequences of different animals are identified by the software and Identified by examining the relevant parts of the chromatogram on the computer screen. You. The difference is that data were obtained for both chains and more than one half of the five horses tested. It is represented as a "DNA polymorphism" only if it is present in several instances. Top panel Indicates "A" homozygotes, the middle panel indicates "AT" heterozygotes, The tier panel shows "T" homozygotes.   Alternatively, polymorphic site discovery can be performed using the strategy shown in FIG. You. In this embodiment, the DNA sequence polymorphism is determined by the pC from unrelated member genomic template. A restriction enzyme obtained by reacting a panel of several restriction enzymes on the product of the R reaction. Identified by comparing the nuclease cleavage profiles. Most preferred Alternatively, each restriction endonuclease used has a four base recognition sequence, Thus, the desired number of cleavages in the amplification product will be possible.   Restriction digestion patterns obtained from genomic DNA can be analyzed using the corresponding plasmid template. Compare directly with the pattern obtained from the obtained PCR product. Such a comparison is , Amplified sequences from genomic DNA and plasmid DNA from equivalent loci Provide an internal control to indicate that This control can also be a repeat sequence or Allows identification of primers that have accidentally amplified a multicopy locus. why Then these are more from the genomic DNA template than from the plasmid template of Because it will generate fragments. III. Methods for genotyping single nucleotide polymorphisms of the invention   In order to identify the polymorphic site "X" of the single nucleotide polymorphism of the present invention, various methods are used. Any of the methods can be used. A preferred method of such identification is to analyze and For each polymorphism that has been identified. Therefore, this appro Is an RFLP method that analyzes band patterns rather than specific sequences of polymorphisms. Significantly different from   A. Amplification-based analysis   Detection of a polymorphic site in a DNA sample is facilitated by using a DNA amplification method. You. Such methods may involve sequences that span the polymorphic site, or a distance between the polymorphic site and the site. Or the sequence located in the proximal or proximal position is specifically increased. That Amplified molecules can be easily detected by gel electrophoresis or other means .   The most preferred way to achieve such amplification is in its double-stranded form. Using a primer pair capable of hybridizing to a proximal sequence that defines a polymorphism Adopt R.   Alternative methods such as “ligase chain reaction” (“LCR”) can be used instead of PCR. (Barany, F.Proc . Natl. Acad. Sci. (USA) 88: 189〜193 (1991) ). LCR indexes specific targets using two pairs of oligonucleotide probes Amplify functionally. The sequence of each oligonucleotide pair is determined by the Select so that it can hybridize to the upper adjacent sequence. Such a hybrid The ligation forms a template-dependent ligase substrate. Like PCR, The product thus obtained serves as a template for the subsequent cycles and provides the desired sequence index. Numerical amplification is obtained.   In accordance with the present invention, the LCR derives proximal and distal sequences on the same strand of the polymorphic site. It can be carried out by using oligonucleotides having each. In one embodiment Designed so that either oligonucleotide contains the actual polymorphic site of the polymorphism Will be done. In such embodiments, the reaction conditions are such that the target molecule is the oligonucleotide. Contain or contain specific nucleotides complementary to polymorphic sites present on nucleotides So that oligonucleotides can be ligated together only if deleted Selection Select.   In another embodiment, the oligonucleotides do not contain polymorphic sites and they When hybridized to offspring, a "gap" will be created (Segev, D . See PCT application WO 90/01069). This gap then has complementary by dNTPs (mediated by DNA polymerase) or by another oligonucleotide Filled with nucleotide pairs. Therefore, at the end of each cycle each single strand Has a complementary strand that can serve as a target in the next cycle, and Numerical amplification is obtained.   "Oligonucleotide Ligation Assay (" OLA ")" (Landegren) , U. et al.Science 2411077-1080 (1988)) have certain similarities to LCR. Can be adapted for use in type analysis. In the OLA protocol, the target Two oligonucleotides designed to hybridize to adjacent sequences of the main strand Use otide. OLA, like LCR, is particularly suitable for detecting point mutations. Only However, unlike LCR, OLA is not an exponential amplification of the target sequence, A "linear" amplification is obtained.   Nickerson, D.A., et al. Have developed a nucleic acid detection system that combines the attributes of PCR and OLA. Say (Nickerson, D.A. et al.Proc . Natl. Acad. Sci. (USA) 87: 8923-8927 (1990)). In this way, exponential amplification of the target DNA is achieved. PCR is used to detect the target DNA, and the target DNA is detected using OLA. Multiple and separate In addition to the need for various processing steps, one of the Problem is that it inherits all the problems associated with PCR and OLA It is.   Two (or more) oligonucleotides are combined into the resulting Ligation in the presence of a nucleic acid having the sequence Thus, a method for amplifying the di-oligonucleotide is also known (Wu, D.Y., et al.). ,Genomics Four: 560 (1989)) and can be easily adapted for the purposes of the present invention. You.   Other known nucleic acid amplification methods, such as transcription-based amplification systems (Malek, L.T. et al. U.S. Patent No. 5,130,238; Davey, C. et al., European Patent Application No. 329,822; Schuster et al., U.S. Patent No. 5,169,766; Miller, H.I. et al., PCT application WO89 /. 06700; Kwoh, D. et al.Proc . Natl. Acad. Sci. (USA) 86: 1173 (1989); G ingeras, TR et al., PCT application WO 88/10315) or isothermal amplification (Walke). r, G.T.Proc . Natl. Acad. Sic. (USA) 89: 392-396 (1992)) Can be   B. Preparation of single-stranded DNA   Direct analysis of SNP sequences in the present invention is also known as "Sanger method". "Dideoxy-mediated chain termination method" (Sanger, F. et al.J . Molec. Biol. 94： 441 (1975) ) Or "chemical decomposition method" (Maxa-Gilbert method) m, A.M.Proc . Natl. Acad. Sci. (USA) 74: 560 (1977)) (see both references) Therefore, any of the methods described above can be used. Zideoki Determine the DNA sequence using either the mediation method or the Maxam-Gilbert method. The method of defining is well known to those skilled in the art. One such method is, for example, Samb rook, J. et al.Molecular Cloning , A Laboratory Manual, 2nd edition,Cold Su Pulling Harbor Press , Cold Spring Harbor, New York (1989) and Zyskind, J.W. et al.Recombinant DNA Laboratory Manual,A Cademic Press New York (1988) (both references are incorporated herein by reference. Which is incorporated herein by reference.   When the nucleic acid sample contains double-stranded DNA (or RNA), or double-stranded nucleic acid amplification When a protocol (such as PCR) is used, one of the two strands is enriched. Double-stranded molecule, preferably so as to obtain a preparation containing only one strand preferentially. It is generally desirable to perform such sequence analysis after processing. However, heat Heat and cool the reaction one or more times using a stable polymerase If so, the present invention does not require the generation of a single-stranded DNA template. By this processing The double-stranded template is separated from its complementary strand, and the subsequent cooling step Can be annealed with the primer. Hybridization with other template strands Competition for the isomerization can be compensated for by repeated cycles of melting-cooling conditions.   The simplest way to produce single-stranded DNA molecules from double-stranded DNA is to use heat Or a modification using either of the alkali treatments. Single-stranded DNA molecules also It can be produced using single-stranded DNA bacteriophage M13 (Messin g, J. et al.Meth . Enzymol. 101: 20 (1983); Sambrook, J. et al.Molecular Cloning . A Laboratory Manual , Cold Spring Harbor Laboratory (See also Les, Cold Spring Harbor, New York (1989).) .   Several alternatives can be used to generate single-stranded DNA molecules. Gyllensten, U. et al.(Proc . Natl. Acad. Sci. (USA) 85: 7652-7656 (1988)) and And Mihovilovic, M. et al. (BioTechniques 7 (1): 14 (1989)) is an asymmetric PCR. The method describes a method in which primers are present at different molar concentrations. Perform a standard "PCR" procedure using. Higuchi, R.G. et al. (Nucl . Acids Res. 17: 586 5 (1985)) illustrates another method for producing single-stranded amplification products. This Method phosphorylates the 5 'end of one strand of the double-stranded amplification product and then 5' → 3 'Preferentially phosphorylated chains to exonucleases (such as T7 exonuclease) The content shall be disassembled.   Other methods also involve phosphorothioate attraction to produce single-stranded DNA molecules. Utilizes the nuclease resistance properties of conductors (Benkovic et al., US Pat. No. 4,521,50) No. 9; Saysers, J.R. et al.Nucl . Acids Res. 16: 791-802 (1988); Eckstein, F. et al.Biochemistry Fifteen: 1685-1691 (1976); Ott, J. et al.Biochemistry 26: 8237〜8241 (1 987)).   On the relative advantages and disadvantages of methods of producing such single-stranded molecules For a discussion, see Nikiforov, T. (U.S. patent application Ser. No. 08 / 005,061 (filed June 1994). (Discarded 24), cited herein for reference).   In the most preferred embodiment, a phosphorothioate derivative is included in the primer. The nucleotide derivative can be introduced at any position of the primer, Preferably introduced at the 5 'end of the primer, most preferably adjacent to each other Will. Preferably, the primer molecule has a complementary region of about 25 nucleotides in length. About 4% to about 100% (compared to all residues), more preferably about 4% Will contain from about 40%, most preferably about 16%, of phosphorothioate residues. . These nucleotides may be introduced at any position of the primer and are adjacent to each other. Contact Or may be interspersed with all or part of the primer.   In one embodiment, the invention is for use with an amplification protocol, such as PCR. Can be. In this embodiment, the established P The above primer can be used without changing the CR protocol The number of phosphorothioate linkages in the primer is about 10 (about half the length of the primer) Minutes). Phosphorothioate linkage beyond primer When PCR conditions are included, PCR conditions are especially It is necessary to adjust with respect to the temperature.   The introduction of such nucleotide derivatives into DNA or RNA is accomplished by DNA It can be performed enzymatically using limerase (Vosberg, HP, et al.Biochemistr y 16: 3633-3640 (1977); Burgers, P.M.J. et al.J. Biol. Chem. 254: 6889〜6893 (1979); Kunkel, T.A., et al., In:Nucleic Acids and Molecular Biology Vol. 2,12 4-135 (Eckstein, F. et al. Eds.), Springer-Veerark, Berlin (1988); Olsen, D.B. et al.Proc . Natl. Acad. Sic. (USA) 87:, 1451-1455 (1990); Gr iep, M.A.Biochemistry 29: 9006-9014 (1990); Saysers, J.R., et al.Nucl . Aci ds Res. 16: 791-802 (1988)). Alternatively, phosphorothioate nucleoti Derivatives can be synthetically introduced into oligonucleotides (Zon, G .;Anti-Canc . Drug Des. 6: 539-568 (1991)).   The primer molecule hybridizes to a complementary target nucleic acid molecule and then preferably Can be extended by a polymerase to form an extension product. Primer Of phosphorothioate nucleotides in the nuclease attack extension products Make it resistant to As noted, phosphorothioate or other suitable Amplification products containing nucleotide derivatives include T7 exonuclease and exonuclease. “Exclusion” (ie, degradation) by “5 ′ → 3 ′” exonuclease such as ) Is substantially resistant and therefore the 5 ′ → 3 ′ exonuclease When it comes to the holothioate residue, the exonuclease binds the nucleic acid molecule. No further decomposition would be possible.   Since the target molecule lacks nuclease resistant residues, the extension product and its template (target When the target is incubated in the presence of 5 ′ → 3 ′ exonuclease, the template strand breaks. Breakage results, whereby preferential production of the desired single strand is achieved.   C. Hybridization of DNA in solution   A preferred method for determining the identity of a polymorphic polymorphic site is nucleic acid hybridization. Including Such hybridization can be performed on a solid phase. Kiruga (Saiki, R.K. et al.,Proc . Natl. Acad. Sci. (USA) 86: 6230〜6234 (198 9); Gilham et al.J . Amer. Chem. Soc. 86: 4982 (1964) and Krembsky et al.,Nucl . Acids Res. Fifteen: 3131-3139 (1987)), and preferably hybridized in a solution. Shin (Berk, A.J. et al.Cell 12: 721-732 (1977); Hood, L.E., et al., In:Molecular B iology of Eukaryotic Cells: A Problems Approach , Menlo Park, Califor Lunia: Benjamin-Cummings (1975); Wetmer, J.G.Hybridization and R enaturation Kinetics of Nucleic Acids . Ann . Rev. Biophys. Bioeng. Five: 33 7-361 (1976); Itakura, K. et al.Ann . Rev. Biochem. 53: 323-356 (1984)).   For application to high capacity testing, it is desirable to use a non-radioactive detection method. Soy sauce Fluorescently labeled or haptenated dideoxynucleotides It is preferred to use The use of biotinylated ddNTPs is based on four of each (3-amino Nopropin-1-yl) nucleoside triphosphate is converted to sulfosuccinimidyl 6- ( It is preferably prepared by reacting (biotinamide) hexanoate. . At that time, the (3-aminopropyn-1-yl) nucleoside 5′-triphosphate is H obbs, by F.W. (J . Org. Chem. 54: 3420-3422 (1989)) and Hobbs, F.W. (US Pat. No. 5,047,519).   D. Analysis of polymorphic sites   1. Polymerase-mediated analysis   The identity of the nucleotides at the polymorphic site of the invention can be determined, for example, by Goelet, P. et al. Variants of the disclosed oligonucleotide-based diagnostic assays for nucleic acid sequence variations (PC T application WO 92/15712, incorporated herein by reference). it can. In particular, the present invention allows simultaneous or near-simultaneous analysis of multiple SNPs Or US Patent Application Ser. No. 08 / 216,538 for ease of reference. To the SNP assay described in US Pat. Things.   To achieve such an advance, the present invention preferably provides the target molecule with a solution in solution. One or more purified interrogates having a predetermined sequence capable of hybridizing Use a solution oligonucleotide. "Interrogation Oligonucleotides "Is" generally refers to the immediate or proximal region of one or more single nucleotide polymorphisms. An oligonucleotide primer having a sequence complementary to a distal sequence.   In a preferred embodiment, one or more sequences having a sequence complementary to a particular region of the target molecule. Use the above method for additional interrogation oligonucleotide primers And prepare it. Preferably, the primer is about 1 complementary to a particular region of the target molecule. It has 2 to 20 bases. These oligonucleotide primers So that the 3 'end of is immediately adjacent to the target nucleotide (eg, SNP) of interest Hybridizes to the target molecule. Preferably, an oligonucleotide primer Are neutral components (eg, poly A, non-basic residues, or other non-specific hybrids). A non-redundant polymer or chemical label). Most preferred In a preferred embodiment, the neutral components are assigned a specific unique length. Primer May or may not include a primer-specific label. I However, in the most preferred embodiment, the oligonucleotide primer is a primer. Includes a mer-specific label.   The interrogation primer is then replaced with one or more single nucleotides. A target DNA molecule having a polymorphism (preferably a genomic DNA molecule) (for each SNP About its immediate 3 'distal sequence is complementary to the interrogation primer ), DNA polymerase and strand terminating nucleotides (or nucleotide analogs) G) Incubate in the presence of the triphosphate derivative. Preferably, such an Incubation is based on dNTPs (ie, dATP, dGTP, dCTP, d TTP) in the total absence of one or more chain-terminating nucleotide triphosphates Conductor (or analog) (eg, ddATP, ddGTP, ddCTP, d a single base of the derivative to the 3 'end of the primer in the presence of Perform under conditions that allow introduction. Unintroduced nucleotide triphosphate present in the reaction solution Is not critical to the reaction, but such unintroduced nucleotides It can be separated by several means. The identity of the introduced nucleotide is Is determined by the nucleotide at the polymorphic site of the polymorphism and is complementary to the nucleotide .   In the present invention, the non-extendable nucleotide is preferably³²P or fluorescent molecule May be labeled. Other labels suitable for the present invention include, but are not limited to: But biotin, iminobiotin, hapten, antigen, cofactor, dinitrophenol Lipoic acid, olefin compounds, detectable polypeptides, high electron density (el ectron dense) molecules, enzymes that can deposit insoluble reaction products. Departure Fluoresceins, loaders include, but are not limited to, Min, Texas Red, FAM, JOE, TAMRA, ROX, HEX, TET , Cy3, Cy3.5, Cy5, Cy5.5, IRD40, IRD41 and BO DIPY. Electron dense indices suitable for the present invention ator) molecules include, but are not limited to, ferritin, Nin and colloidal gold. A detectable polypeptide is detectable Specifically conjugates a polypeptide to a second polypeptide covalently linked to an indicator molecule It may be indirectly detectable by forming a body. In such an aspect The detectable polypeptide is a group consisting of avidin and streptavidin Preferably, the second polypeptide is biotin and iminobioti Preferably, it is selected from the group consisting of   In a preferred embodiment, the resulting extended primer is placed on a suitable matrix. Separate for analysis. To separate extended primers for analysis Any of a number of methods can be used. One such method is But not limited to mass spectrometry, (oligonucleotides Array hybridization) flow cytometry, HPLC, FPLC, Size exclusion chromatography, affinity chromatography, gel electricity Electrophoresis and the like. Preferably, the extended primer is separated under denaturing conditions. However, denaturing conditions are not required for effective separation.   Non-extendable nucleotides are synthetic or can be introduced in a template-dependent manner. Refers to naturally occurring nucleotide analogs. Suitable for use in the present invention Synthetic or naturally occurring nucleotide analogs Non-cyclic ribose nucleotide analogs, substituted ribose nucleosides Tide analogs, and modified ribose nucleotide analogs. Synthetic The nucleotide analog is preferably a fructose-based nucleotide analog. Chemically modified probes that retain the ability to specifically base pair with natural nucleotides Phosphorus, a chemical modification that retains the ability to specifically base pair with natural nucleotides Retains the ability to base pair specifically with pyrimidines and natural nucleotides Selected from the group consisting of all compounds.   In the most preferred embodiment, the separation of the extended primer Denaturing size separation matrices, such as standard sequencing gels with mid concentrations Do above. In this embodiment, an interrogation system comprising a specific unique length of 5 'tail is provided. Use primers. The extended primer is a specific unique length of 5 'tape. Is separated based on the file. In a preferred embodiment, the labeled chain terminating nucleotide ( Or nucleotide analogs), but this embodiment also uses It is also directed to the gating primer. In one sub-embodiment, A chain terminating nucleotide (ie, dideoxynucleotide) is used. Another subform In one embodiment, one or more strand terminators are labeled with only a single strand terminating nucleotide. Use nucleotides.   In another preferred embodiment, it will hybridize to a complementary sequence located on the solid phase. Interrogation primers containing unique and specific sequences are used. This aspect Now, expose the interrogation primer to the placed capture primer and fix it. By detecting labeled bases based on their position on the phase capture primer array, Separated by identifying single nucleotide polymorphisms.   In another embodiment, the resulting extended primer has an appropriate affinity Separated using separation method. Such affinity separation methods are generally Ter-ligand method (eg, avidin-streptavidin, etc.), monochrome Related to the local antibody method. In this embodiment, the 5 'end of each primer is Binds to the unique ligand present. In this embodiment, Put the seven puters on a solid surface (ie, beads, columns, dipsticks, microphones). It is preferably immobilized on a titer plate. Ligand labeled plies The mer is separated by exposing the reaction mixture to the corresponding receptor. Incidentally The identity of each single nucleotide polymorphism can be determined using the methods described above.   In other embodiments, residues of unique sizes (eg, BSA, lysozyme, egg alga) Using primers (covalently or otherwise) conjugated to (eg, cumin) . In this embodiment, unique size primers are appropriately size exclusion chromatographic. Pass the primer through a feed column and use the above method to make each single nucleotide polymorphism. Are determined by determining the identity of   In the present invention, a single reaction can be performed by using the above methods in combination. The number of SNPs that can be identified can be increased.   Labels suitable for use in the present invention include, but are not limited to: Not available, but enzymes (β-galactosidase, luciferase, etc.), radioisotopes ( That is,³²P,¹³C,^ThreeH), fluorescent residues (ie, fluorescein, And chromogens. Primers can be labeled directly Or to a separate ligand, labeled or unlabeled You can also. The ligand molecule is covalently bound to the oligonucleotide primer, Ionic interactions, non-specific adsorption, or specific but non-covalent ligands It can be bound by receptor interaction.   A ligand is generally a protein of interest for which there is a corresponding distinct receptor Or a chemical substance. Suitable ligands for use in the present invention include these Hapten, antigen, cofactor, biotin, iminobioti , Dinitrophenol, riboacid, olefin compound, oligonucleotide, phase Tags designed to specifically hybridize to complementary oligonucleotides Protein nucleic acid ("PNA") sequences and PNA sequences functioning as receptors Is mentioned. Other ligands suitable for use in the present invention include, but are not limited to: Antibodies, enzymes, polypeptides, streptavidin and Avidin is mentioned. In one embodiment, the ligand is a detectably labeled polypeptide. A complex can be formed by binding to the polypeptide. Used in the present invention Suitable detectable labels include, but are not limited to, , Enzymes capable of depositing insoluble reaction products, streptavidin and avidin Is mentioned. Preferably, the detectably labeled polypeptide is produced randomly. Select from the resulting polypeptide library.   A receptor is generally a defined protein or protein on which the corresponding ligand is present. Refers to a chemical substance. Receptors suitable for use in the present invention include, but are not limited to: Antigens, cofactors, biotin, iminobiotin, dinitroph Enols, lipoic acids, olefinic compounds, oligonucleotides, complementary oligonucleotides A PNA sequence designed to specifically hybridize to a nucleotide; and And a PNA sequence that functions as a ligand. In one embodiment, the reception The putter forms a complex by binding to a detectably labeled polypeptide. Can be Suitable detectable labels for use in the present invention include: Antibodies, enzymes that can deposit insoluble reaction products, Leptoavidin and avidin are mentioned. Preferably, it is detectably labeled The polypeptide is selected from a randomly generated polypeptide library. The receptor may be bound to a matrix. Binding receptor to matrix Suitable methods for this include, but are not limited to, covalent bonding, Interaction, nonspecific adsorption, or specific but noncovalent ligand-receptor Putter interaction. Ligand-receptor suitable for use in the present invention One is, but not limited to, a complementary hybridizing nucleus. Acids, complementary hybridizing PNAs, and other complementary synthetic nucleic acid analogs Is mentioned.   In a preferred embodiment, the extended primer is separated prior to detection. It can also be used to identify SNPs without separating primers . In this embodiment, (either by labeling the oligonucleotide or by the oligonucleotide Nucleotide interrogation primer (by not labeling the tide) At least one chain-terminating nucleotide triphosphate so that the addition to Label conductors uniquely. Therefore, for example, three different SNPs were When three primers were used in the presence of labeled ddATP for The introduction of the label indicates that one of the SNPs is T.   Identification of primers by primer-specific labeling and introduced nucleotides , Enables genotyping of target molecules. Therefore, the nucleotide at the polymorphic site is Whichever of the set of labeled nucleotides is By assaying whether it was introduced at the 3 'end of the oligonucleotide Is done. Non-extendable nucleotides or nucleotide analogs can be Or it can be identified by any of the chemical methods. However, preferred physical Or chemical means, polarization spectroscopy, mass spectroscopy, infrared spectroscopy, ultraviolet spectroscopy It is selected from the group consisting of analysis, visible light spectroscopy or NMR spectroscopy.   Although the present invention is directed to a method for identifying a plurality of SNPs in a single reaction, The present invention can also confirm the identity of multiple SNPs in a single reaction. In this embodiment, each SNP for both the plus and minus strands of the target nucleic acid Identity is determined as described above. The sequence is pro- cessed for each SNP analyzed. Identified when the ras and minus strands are complementary.   2. Polymerase / ligase-mediated analysis   In another embodiment, the nucleotide at the polymorphic site is a polymerase / ligase mediated method Is identified using As in the above embodiment, multiple SNPs are detected by the same reaction To use multiple oligonucleotide primers simultaneously.   Oligonucleotides complementary to the immediate non-mutated sequence immediately 3 'of the SNP, as in the above embodiment. Use a reotide primer. Complementary to the 5 'proximal sequence of the polymorphism to be analyzed but not Second oligonucleotide that cannot hybridize to the oligonucleotide primer Use   These oligonucleotides are converted to DNA containing single nucleotide polymorphisms to be analyzed. And in the presence of at least one 2 ', 5'-deoxynucleotide triphosphate Incubate. This incubation reaction is further performed with DNA Polymerase. And DNA ligase.   Therefore, both oligonucleotides have the same single nucleotide polymorphism to be analyzed. It can hybridize to a chain. Due to sequence considerations, these two oligos Nucleotides are located proximal to the SNP flanking the polymorphic site (X). And to the distal sequence; hence, these hybrids The modified oligonucleotide is a single nucleotide "gis" at the exact location of the polymorphic site "Caps".   Polymerase and 2 ', 5'-deoxynucleotide triphosphate complementary to (X) The presence of an acid extends the complementary 2 ', 5'-deoxynucleotide triphosphate Primer and a hybridized oligonucleotide complementary to the distal sequence. Ligation is possible and 2 ', 5'-deoxy complementary to polymorphic nucleotides Synonucleotide triphosphates generate a ligatable substrate.   Then, the identity of the polymorphic site opposite the "gap" can be determined by several means. It can be determined by any of them. In a preferred embodiment, the 2 ', 5'- The deoxynucleotide triphosphate is labeled, and its detection complements the polymorphic site. Target nucleotides. Several different 2 ', 5'-deoxynucleotide tris Phosphoric acid may be present and each is labeled differently. Alternatively, separate anti And a different 2 ', 5'-deoxynucleotide triphosphate is used in each reaction. In another sub-embodiment, the 2 ′, 5′-deoxynucleotide triphosphate is unlabeled, Label the two soluble oligonucleotides. Perform separate reactions, each with a different 2 ', 5'-deoxynucleotide triphosphate is used.   The above embodiment details a polymerase / ligase mediated method for detection of a single polymorphic site. As mentioned, this method provides multiple unique options for the detection of multiple polymorphic sites. It is generally understood that simultaneous use of lignonucleotide primers can be employed Will.   E. FIG. Signal amplification   Nucleic acid hybridization detection assay sensitivity does not report detection to observer It can be enhanced by changing the way it signals. Therefore For example, assay sensitivity can be increased by using detectably labeled reagents. Can be. A wide range of such signal amplification methods have been designed for this purpose. Have been. Kourilsky et al. (U.S. Pat. No. 4,581,333) report detection using enzyme labels. It describes increasing the sensitivity of the assay. Fluorescent labels (Albarella et al., EP 14491 No. 4), chemical labels (Sheldon III et al., US Pat. No. 4,582,789; Albarella et al., US Patent No. 4,563,417), modified bases (Miyoshi et al., EP 119448), etc. It has been used in an effort to improve the efficiency of observing solicitation.   Automatic or semi-automatic detection of the identity of the introduced nucleotide using an appropriate detector Using fluorescent or chromogenic (especially enzyme) labels as can be determined by Is preferred. IV. Use of SNP genotyping in methods of genetic analysis   A. General considerations for using single nucleotide polymorphisms in gene analysis   The usefulness of the polymorphic site of the present invention is that such a site can Can be used to predict the statistical probability of having the same allele Based on what you can do.   Statistical analysis of SNPs can be used for any of a variety of purposes. If a particular individual has been previously tested, such a test may be -Prints, which determine the identity of a particular individual. Can be used to   If the putative parent or both parents of a particular individual are tested, The law determines whether a particular animal is a descendant of such parent or both parents. Can be used to determine likelihood. Hence, the detection and analysis of SNPs Is the paternity of a male (a child of a particular child) Selected females, which excludes the father's paternity, etc.) or has certain individuals (E.g., to determine the probability of being a descendant of a particular child and a selected mother) Can be.   As shown below, the present invention allows the construction of a genetic map of a target species. Soy sauce Of certain animals (or plants) to such genetic diseases, conditions, or attributes To predict a predisposition, a particular array of polymorphisms identified by the method of the invention is identified by a particular array. Can be associated with attributes. As used herein, “characteristics” refers to “genetic diseases”, It is intended to encompass "conditions" or "characteristics." "Genetic disease" Can be detected by mutation, whether detectable or asymptomatic. Refers to the pathological condition caused. "Condition" refers to a characteristic (asthma, weak bone, blindness) Ulcers, cancer, diseases of the heart or cardiovascular system, skeletal-muscular defects, etc.) . "Characteristics" are attributes that confer economic value on a plant or animal. Examples of characteristics and Examples include longevity, speed, durability, aging speed, and reproductive ability.   B. Identification and parental confirmation   The most useful measurements to determine the efficacy of the identification and paternity test system are (i) "Probability of identity (p (ID))" and (ii) "exclusion" (Probability of exclusion) (p (exc)). " p (ID) is 2 Two random individuals will have the same genotype for a given polymorphic marker To calculate the likelihood. p (exc) is, for a given polymorphic marker, A random male is a father in an average paternity case where mother identity is not an issue. Calculates the likelihood of having a genotype that is incompatible with being a parent You. A single locus (a locus with multiple alleles, such as a major histocompatibility region) Does not provide an appropriate statistically-reliable test for paternity testing Since rare, the desired test preferably parallels multiple unlinked loci. Will be measured. Cumulative probability of identity or non-identity, and cumulative paternity exclusion Probability is calculated by multiplying the probability given by each locus by Decided for the locator test.   The most interesting statistical measures are (i) the cumulative probability of non-identity (cump (nonID)) ) And (ii) the cumulative probability of paternity exclusion (cump (exc)).   The equations used to calculate these probability values are shown below. For simplicity, these Are first given to two allele loci, one allele is called type A, and the other is It is called B type. Four genotypes are possible in such a model: AA, AB , BA and BB (AB and BA types cannot be distinguished biochemically ). The frequency of alleles is the number of A's found in the haploid genome (f (A), The frequency of B (f (B), the frequency of B is “q” (where q = 1 −p)). Predetermined at a given genotype locus The genotype probabilities are shown below: Homozygote: p (AA) = p^Two Single heterozygote: p (AB) = p (BA) = pq = p (1-p) Both heterozygotes: p (AB + BA) = 2pq = 2p (1-p) Homozygote: p (BB) = q^Two= (1-p)^Two   Probability of identity at a locus (ie, randomly picking from a population) The probability that the two individuals will have the same genotype at a given locus) , Equation: p (ID) = (p^Two)^Two+ (2pq)^Two+ (Q^Two)^Two Given by   Therefore, the cumulative probability of identity for n loci is given by the equation: Given by   Cumulative probability of non-identity for n loci (ie, random Probability that two individuals picked at different times will differ at one or more loci ) Is the equation: cum p (nonID) = 1-cum p (ID) Given by   Probability of paternity elimination (random males question maternal identity for a given locus) Genes that are incompatible with being male parents in an unnamed average paternity case Indicating the probability of having a type) is given by the equation: p (exc) = pq (1-pq) Given by   Probability of non-exclusion (random male at given locus is average paternity case Represents the probability of not being biochemically excluded as a male parent in the equation): p (non-exc) = 1-p (exc) Given by   Therefore, the non-excluded cumulative probability (the value obtained when using n loci Is) It is.   Cumulative probability of exclusion (using a panel of n loci, random male Biochemically excluded as a male parent in an untitled average paternity case Is the probability that cum p (exc) = 1-cump (non-exc) Given by   These calculations extend to any number of alleles at a given locus it can. For example, three alleles each having a frequency p, q and r in the population The probability of identity p (ID) for the rel system is equal to the sum of the squares of the genotype frequencies : p (ID) = p^Four+ (2pq)^Two+ (2qr)^Two+ (2pr)^Two+ R^Four+ Q^Four   Similarly, the probability of exclusion for the three allele system is p (exc) = pq (1-pq) + qr (1-qr) + pr (1-pr) + 3pqr (1-pqr) Given by   At the n allele locus, p (ID) and And p (exc).   FIG. 3 and FIG. 4 show that both the number and type of loci used It shows how nonID) and cum p (exc) increase. Using 3 allele system It can be seen that better discriminative power can be achieved with fewer markers. 3 and 4, triangles increase the number of loci with two alleles The common allele is the frequency of p = 0.79. Exists in degrees. X in FIGS. 3 and 4 means p = 0.5, q = 0.34 and r A similar analysis is shown for an increase in the number of triallelic loci at = 0.15.   However, any loci with 2, 3 or more alleles should be selected. The choice is largely influenced by the biochemical considerations described above. Polymorphism analysis tests Can be designed to score any number of alleles at a given locus . If alleles are scored using gel electrophoresis, each allele should be It must be easy to analyze by movement. With multiple allele families Are often small, so human DNs using multiple allele families The A test includes a statistical correction for misidentification of the allele. In addition, multiple Although the appearance of rare alleles from the rel system is very informative, Rareness makes accurate measurement of their frequency in a population extremely difficult. I have. To correct errors in these frequency evaluations when using rare alleles , Statistical analysis of this data provides a measure of the cumulative effect of uncertainty in these frequency assessments. To Must include. The use of these multiple allelic systems also New or rare alleles in the population will be discovered during screening Increase prospects. The completeness of the genetic data collected so far is a novel allele It will be revised empirically to reflect the discovery of.   With these considerations in mind, using loci with multiple alleles is Although it may provide some short-term benefits (fewer loci (I) because it does not need to be screened), (i) it is represented more frequently, and (ii) A locus with fewer alleles that is clearly easier to measure It is preferable to perform polymorphism analysis using the same. In this type of study, the more polymorphic Can achieve the same discrimination as a locus-based test, but with the same total number of alleles Must be recovered from a series of unlinked loci.   G. FIG. Gene mapping and genetic trait analysis using SNP   Detected in a set of individuals of the same species (human, horse, etc.) or closely related species Polymorphisms indicate that the presence or absence of a particular polymorphism correlates with a particular attribute Can be analyzed to determine whether or not.   To perform such polymorphism analysis, a set of polymorphisms (ie, a “polymorphism array”) Is determined for a set of individuals. How many of these individuals Indicate specific attributes, and some are mutually exclusive attributes (eg, horses). In terms of brittle and non-brittle bones; blindness and non-blindness beginning in maturity; asthma Predisposition and absence of such predisposition). By the way, Examining the alleles of each polymorphism in the set to determine the presence or absence of a particular allele. Determine whether it is associated with a particular attribute to taste. Such a correlation Defines the genetic map of the individual species. Arrays that do not separate randomly Are used to predict the probability that a particular animal will develop the property be able to. For example, members of a species where a particular polymorphic allele exhibits a cardiovascular condition Is present in only 20% of the species, certain members of that species, including the allele, Would have a 20% probability of indicating such a cardiovascular condition. To point out In addition, the predictive power of this analysis depends on the association between a particular polymorphic allele and a particular property. Increases with the degree of Similarly, the predictive power of this analysis is Polymorphism It can be increased by analyzing alleles at the locus simultaneously. the above In an example, the second polymorphic allele is also present in 20% of members exhibiting a cardiovascular condition. All members evaluated to present such a cardiovascular condition should And a particular combination of alleles of the second polymorphism, and therefore Certain members, including both rels, have a very high probability of exhibiting a cardiovascular condition Will.   Detection of multiple polymorphic sites determines the frequency with which such sites are independently segregated in the population. To be able to For example, if the two polymorphic sites separate randomly , They are on separate chromosomes or separated from each other on the same chromosome I do. Conversely, two polymorphic sites that are simultaneously inherited at a significant frequency are located on the same chromosome. Linked to each other. Thus, analysis of the frequency of segregation is Enable the establishment of a map. Therefore, the present invention maps the genomes of plants and animals. To provide a means for performing   The resolution of a genetic map is proportional to the number of markers it contains. The method of the present invention Because any number of polymorphic sites can be used to isolate It can be used to create maps with resolution.   Sequencing polymorphic sites greatly enhances their utility in gene mapping . Such sequences "walk" the chromosome, thereby identifying new marker sites Oligonucleotide primers and probes that can be used to (Bender, W. et al.,J . Supra. Molec. Struc. 10 (Addendum) : 32 (1979); Chinault, A.C., et al.Gene Five: 111-126 (1979); Clarke L .; La ,Nature 287: 504-509 (1980)).   The resolution of the map can be determined by analyzing the polymorphism analysis in plants or Is further enhanced by combining with data on the phenotype of other attributes of the animal. Can be Therefore, certain polymorphisms segregate with brown hair color If the polymorphism maps to a locus near the hair color gene or genes, Is performed. Similarly, increasing the resolution of genetic maps using biochemical data Can be. In this embodiment, the biochemical determination (serotype, isotype, etc.) It is examined to determine if it segregates with any of the polymorphic sites. That's it Unmaps can be used, for example, to identify new gene sequences or to cause disease-causing mutations. Can be used to identify   Indeed, the identification of the SNPs of the present invention has led to the discovery of a novel residue located on either side of the SNP. To isolate or sequence the gene sequence, the complementary oligonucleotide is Enables use as a primer in CR or other reactions. The present invention Include such novel gene sequences. Using such primers Thus, genomic sequences that can be clonally isolated are transcribed into RNA, Can be translated as quality. The present invention also relates to such proteins, And antibodies and other binding molecules capable of binding to such proteins.   The present invention is described below with respect to two of its aspects, namely horses and humans. explain. However, the basic tenets of genetics can be applied regardless of species So such an explanation is equally applicable to any other species . Therefore, those skilled in the art will recognize that any other species may be Only need to isolate the SNPs and thereby perform the genetic analysis of the present invention. .   As noted above, LOD scoring is useful for tracking the inheritance of genetic traits. Enables the use of RFLP both for constructing genetic maps of species and (Lander, S. et al.,Proc . Natl. Acad. Sci. (USA) 83: 7353 ~ 7357 (1986); Lander, S. et al.Proc . Natl. Acad. Sci. (USA) 84: 2363〜2367 (1 987); Donis-Keller, H. et al.Cell 51: 319-337 (1987); Lander, S. et al.Genetics 1 twenty one : 185-199 (1989)). Such methods are easily adapted for use with the polymorphisms of the invention. Can be combined. In fact, such polymorphisms are not Better than R. Easily create dense genetic maps for SNP frequencies be able to. In addition, as noted above, the polymorphisms of the present invention are typical VNT More stable than R-type polymorphism.   Because the polymorphisms of the present invention contain direct genomic sequence information, Can be divided. Analysis must be gel-based for RFLP or STR dependence maps This entails obtaining an electrophoretic profile of the DNA of the target animal. In addition to gel-based methods, polymorphism (SNP) analysis can also be performed using spectroscopy. And can be easily automated to facilitate the analysis of large numbers of target animals. Wear.   As described above, since the present invention has been generally described, the following is the isolation and analysis of equine polymorphism. The present invention may be better understood with reference to the examples. These practices The examples are illustrative and are not intended to limit the invention.                                 Example 1          Analysis of multiple SNPs by polyacrylamide gel electrophoresis   In this example for the detection of multiple single nucleotide polymorphisms, the single-stranded DNA template Was probed with three interrogation primers. Primer # 1 has a 5 base T-tail, primer # 2 has a 10 base T-tail, Primer # 3 has a 15 base T-tail.   To obtain a single-stranded template, one of two methods was used. First, Niki forov, T .; (US Patent Application No. 08 / 005,061 (application abandoned June 24, 1996) ), Including 4 phosphorothioate-nucleotide derivatives, as taught by Primers are used to mediate amplification. Alternatively, the second round of PCR may be This can be done using "symmetric" primer concentrations. The product of the first reaction is transferred to the second In the reaction, the dilution is 1/1000. One of the primers in the second round has a 2M mark Use at a sub-concentration, the other at a concentration of 0.08M. Under these conditions, single-stranded molecules It is synthesized during the reaction.   The primer mixture is hybridized to the single-stranded target template and the DNA polymerase Initiate a single base extension reaction using a modified and four modified non-extendable nucleotides. Rub. Only one modified non-extendable nucleotide for each four reaction tubes Preferably³²Label with P or fluorescent molecule. Then, the obtained extended primer Are separated for analysis on a standard 12% sequencing gel. Like this To determine the electrophoretic mobility of each primer and the identity of labeled non-extendable nucleotides. It is possible to determine the identity of the SNP corresponding to each primer based on the You.                                 Example 2           Analysis of multiple SNPs by size exclusion chromatography   Primers 1, 2, 3 and 4 were replaced with BSA, egg albumin and lysozyme, respectively. And covalently bound to CIP. Four interrogations of single-stranded DNA template Probe with primers, DNA polymerase and four modified non-extendable A single base extension reaction using nucleotides is caused. Preferably each non-extensible The nucleotides are uniquely and separately labeled.   Subsequently, the resulting extended primer is applied to a suitable size exclusion column (eg, , Sephadex, Sepharose, etc.) and separate the eluate (eg, Analysis (by a titration counter) to determine the identity of the introduced nucleotides. Set.                                 Example 3                Analysis of multiple SNPs using affinity method   In this example, for example, the method disclosed by Chu et al.Nucleic Acids Res . 16 : 3671-3691 (1988), which is incorporated herein by reference). Alternatively, the protein affinity ligand can be interrogated with an oligonucleotide. Covalently bound to Then, the affinity ligand-interrogation probe The primer complex is hybridized to the target nucleic acid molecule and the single base extension is performed for four times. Of differently labeled dideoxynucleotide species (ddA, ddT, ddC and ddG). If desired, less species of dideoxynucleated Reotide may be used.   The corresponding monoclonal antibody against the peptide or protein Immobilize on an interplate (Nunc). Buffer each monoclonal antibody at room temperature In a microtiter plate. Then, plate the TNTw solution Wash three times to remove excess unbound protein.   The extended primer solution is then added to each well over about 30 minutes. Not yet The bound primer is removed by extensive washing with a TNTw solution. Table 3 shows such facts. Shows the results expected from the experiment. Example 4             Analysis of multiple SNPs without separating oligonucleotides   Multiple single nucleotide polymorphisms without separating oligonucleotide probes Can also be identified. In such experiments, each dideoxynucleotide Preferably, the triphosphate is uniquely labeled.   So, for example, ddATP³²Labeled with P, ddGTP^ThreeLabel with H , DdCTP is³⁵Labeled with S, ddTTP is¹²⁵I can be labeled. table No. 3 hybridizes six oligonucleotides to the preparation containing the nucleic acid of interest. The hypothetical results obtained from soybean and the results of a single base extension reaction are shown. In such experiments, unintroduced ddNTPs could be used in various ways (eg, Any of the appropriate spin columns (ie, CentriSep spin columns) Can be used to separate from the extended probe.   The introduced labeled dideoxynucleotide triphosphate is then scintillated. Detection using a counter. Because each isotope has a separate emission spectrum The scintillation counter allows multiple single nuclei without the need for purification procedures. The identity of the leotide polymorphism can be determined.   As shown in Table 4, the introduced dideoxynuclease complementary to the single nucleotide polymorphism was The identification of the nucleotides was determined by T, G, A, T, C, It is clear that G. Ambiguity is changing primer combinations Can be determined by                                 Example 5           Analysis of multiple SNPs by oligonucleotide array separation   Primers 1, 2, 3 and 4 were used in addition to the sequence complementary to the template DNA, A unique arrangement for subsequent hybridization to the capture oligonucleotide on the surface Contains columns. Probe single-stranded DNA template with 4 interrogation primers Using DNA polymerase and four modified non-extendable nucleotides. Initiate a single base extension reaction. Preferably each non-extendable nucleotide is unique And label separately.   The obtained extended primer is then applied to the surface of the oligonucleotide array. Apply. This array comprises four separate and spatially separated distinct capture oligonucleotides. Consisting of nucleotides, each oligonucleotide is an interrogation primer Complementary to one and above unique sequence. Each interrogation primer is Effective by hybridization to capture primer bound to corresponding surface Separated. The identity of the labeled nucleotide is then determined by a suitable method.   Although the invention has been described with reference to specific embodiments thereof, it is possible to further modify it. This application is generally based on the principles of the present invention, and As is known and practiced in the art, or as described above and in the appended claims. Include those that depart from the disclosure of the invention as applicable to essential features therein. And is intended to cover any modifications, uses, or adaptations of the invention. It will be understood.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, L S, MW, SD, SZ, UG, ZW), EA (AM, AZ , BY, KG, KZ, MD, RU, TJ, TM), AL , AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, E E, ES, FI, GB, GE, GH, GM, GW, HU , ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, M D, MG, MK, MN, MW, MX, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, V N, YU, ZW (72) Inventor Head, Stephen             Han, United States 21074 Maryland             Pstead, South Main Street             808 (72) Inventor Gorlet, Philip             United States 21136 Lee, Maryland             Startown, Millender Mill Road             4000th (72) Inventor Voice-Jachino, Michael Tee             United States 21048 Philly, Maryland             Nksburg, Naina Road 3811

Claims (1)

[Claims]   1.1. A method for identifying one or more single polymorphisms in a single reaction , Process: (A) one or more identifiable interrogation oligonucleotide probes The primer is hybridized to one or more target nucleic acid molecules, wherein each Oligonucleotide primers are of interest where the 3 'end of each primer is of interest. Of each target nucleic acid molecule so that it is immediately adjacent to a constant and unique target nucleotide Complementary to specific and unique regions; (B) Each interrogation oligonucleotide is treated with a template-dependent polymerase. Extension, wherein the extension comprises one or more non-extendable nucleotides or Occurs in the presence of a nucleotide analog species; (C) For each interrogation primer used, such a primer Of non-extendable nucleotides (or nucleotide analogs) introduced into By determining the identity, the identity of each nucleotide of interest is determined. The identified non-extendable nucleotide or nucleotide analog Complementary to the target nucleotide of the primer A method that includes   2. Each interrogation oligonucleotide primer contains a 5 'tail The 5 'tail is of a specific and unique length or each interrogation primer A neutral component having other properties used to identify or separate Item 1. The method according to Item 1.   3. 2. The method of claim 1, wherein said hybridization step occurs in a solution. .   4. Identifying the non-extendable nucleotide by a physical or chemical method The method of claim 1.   5. The physical method or the chemical method is used for polarization spectroscopy, mass spectroscopy, infrared spectroscopy. Select from the group consisting of optical analysis, ultraviolet spectroscopy, visible light spectroscopy or NMR spectroscopy. 5. The method according to claim 4, wherein the method is performed.   6. Further steps: (D) separating the extended primer on a suitable matrix The method of claim 1, comprising:   7. 7. The method of claim 6, wherein said matrix is a size separation matrix. .   8. The method of claim 7, wherein the size separation matrix is a sequencing gel. The method described.   9. The sequencing gel comprises about 4% to about 20% polyacrylamide The method of claim 8.   10. 8. The method of claim 7, wherein the size separation matrix is a size exclusion column. the method of.   11. A suitable receptor molecule is bound to the matrix and the receptor The appropriate ligand molecule corresponding to the oligonucleotide is bound to the oligonucleotide primer. 7. The method of claim 6, wherein the method is performed.   12. The matrix contains beads, columns, dipsticks, Claims selected from the group consisting of interplates and glass slides Item 12. The method according to Item 11.   13. 2. The method of claim 1, wherein said non-extendable nucleotide is ddNTP. .   14． 14. The ddNTP is fluorescently or chemically labeled. The method described in.   15. 14. The method according to claim 13, wherein said ddNTP is biotinylated.   16. 2. The method of claim 1, wherein said target molecule is a nucleic acid molecule.   17． 17. The method according to claim 16, wherein said nucleic acid molecule is a DNA molecule.   18. 18. The method according to claim 17, wherein said DNA molecule is genomic DNA.   19. 18. The method according to claim 17, wherein said DNA molecule is double-stranded DNA.   20. 18. The method according to claim 17, wherein said DNA molecule is single-stranded DNA.   21. 17. The method of claim 16, wherein said nucleic acid molecule is an RNA molecule.   22. A method for characterizing a target DNA, comprising: (A) one or more identifiable interrogation oligonucleotide probes The primer is hybridized to one or more target nucleic acid molecules, wherein each Oligonucleotide primers are of interest where the 3 'end of each primer is of interest. Of each target nucleic acid molecule so that it is immediately adjacent to a constant and unique target nucleotide Complementary to specific and unique regions; (B) Each interrogation oligonucleotide is treated with a template-dependent polymerase. Extension, wherein the extension occurs in the presence of more than one non-extendable nucleotide species. Happen; (C) separating the extended primers on a suitable matrix; (D) For each interrogation primer used, such a primer Of interest by determining the identity of the non-extendable nucleotides introduced into the Examining each nucleotide that leans, wherein the identified non-extendable nucleotides Is complementary to the target nucleotide of the primer; And (E) converting the interested nucleotides of interest to those of a reference nucleic acid molecule. Compared to the corresponding nucleotide, the nucleotide of interest at each site Determine if they contain the same single nucleotide A method that includes:   23. 3. The method of claim 2, wherein the characterization identifies a property of the target DNA molecule. 3. The method according to 2.   24. 24. The method of claim 23, wherein said characteristic is a genetic disease.   25. 24. The method of claim 23, wherein said attribute is a genetic condition.   26. The size separation matrix is from Sepharose and Sephadex The method of claim 7, wherein the method is selected from the group consisting of:   27. The ligand is a hapten, antigen, cofactor, biotin and iminobioti. 12. The method of claim 11, wherein the method is selected from the group consisting of:   28. The ligand is a dinitrophenol, a lipoic acid and an olefin compound. 12. The method of claim 11, wherein the method is selected from the group consisting of:   29. The ligand specifically hybridizes to a complementary oligonucleotide. Unique and specific oligonucleotides, complementary oligonucleotides PNA sequences and receptors designed to specifically hybridize to otide 2. The protein according to claim 1, which is selected from the group consisting of PNA sequences functioning as putters. 2. The method according to 1.   30. The ligand may be an antibody, enzyme, polypeptide, streptavidin and antigen. 12. The method of claim 11, wherein the method is selected from the group consisting of vidin.   31. The ligand is conjugated by binding to a detectable polypeptide. The method of claim 11, which can form   32. The detectable polypeptide may deposit antibodies, insoluble reaction products Enzymes, selected from the group consisting of streptavidin and avidin, 31. The method according to claim 30.   33. The detectable polypeptide is a polypeptide library randomly generated. 31. The method of claim 30, wherein the method is selected from a rally.   34. The receptor is a hapten, antigen, cofactor, biotin and iminobio 12. The method of claim 11, wherein the method is selected from the group consisting of chin.   35. The receptor is a dinitrophenol, a lipoic acid and an olefin compound The method of claim 11, wherein the method is selected from the group consisting of:   36. The receptor specifically hybridizes to a complementary oligonucleotide Unique and specific oligonucleotides, complementary oligonucleotides PNA sequences designed to specifically hybridize to Claims 1. It is selected from the group consisting of PNA sequences that function as sceptors. 12. The method according to 11.   37. The receptor is conjugated by binding to a detectable polypeptide. 12. The method of claim 11, wherein the method is capable of forming a body.   38. The detectable polypeptide may deposit antibodies, insoluble reaction products Enzymes, selected from the group consisting of streptavidin and avidin, 38. The method of claim 37.   39. The detectable polypeptide is a polypeptide library randomly generated. 38. The method of claim 37, wherein the method is selected from a rally.   40. The receptor molecule binds to the matrix via covalent bonds, ionic interactions, Non-specific adsorption and specific but non-covalent ligand-receptor interactions 12. The method of claim 11, wherein the methods are combined by a method selected from the group consisting of:   41. The ligand-receptor is selected from complementary hybridizing nucleic acids. 41. The method of claim 40, wherein   42. Hybridizing PNAs and the like wherein the ligand-receptor is complementary 41. The method according to claim 40, which is selected from the group consisting of: Law.   43. The ligand molecule is covalently attached to the oligonucleotide primer, Interaction, non-specific adsorption, and specific but non-covalent ligand-receptor 12. The method according to claim 11, wherein the binding is performed by a method selected from the group consisting of Putter interactions. The method described.   44. The ligand-receptor is selected from complementary hybridizing nucleic acids. 44. The method of claim 43.   45. Hybridizing PNAs and the like wherein the ligand-receptor is complementary 44. The method according to claim 43, wherein the compound is selected from the group consisting of: Law.   46. The non-extendable nucleotide is introduced by a template-dependent polymerase. 2. The method of claim 1, wherein the compound is a synthetic or naturally occurring nucleotide analog. The method described.   47. The synthetic or naturally-occurring nucleotide analog is an acyclic ribose. , Substituted nucleotide analogs, and modified ribose nucleotide analogs 47. The method of claim 46, wherein the method is selected from the group consisting of:   48. The synthetic nucleotide analog is a fructose-based nucleotide 47. The method of claim 46, wherein the method is selected from the group consisting of Nalog.   49. The synthetic nucleotide analog is specific for a naturally occurring nucleotide. From chemically modified purines or pyrimidines that retain the ability to base pair with 47. The method of claim 46, wherein the method is selected from the group consisting of:   50. The synthetic nucleotide analog is specific for a naturally occurring nucleotide. Selected from the group consisting of compounds that retain the ability to base pair with 47. The method of claim 46.   51. The non-extendable nucleotide is fluorescently labeled or chemically labeled The method of claim 1, wherein the method is performed.   52. The non-extendable nucleotide is labeled with biotin or iminobiotin The method of claim 1, wherein the method is performed.   53. The non-extendable nucleotide is labeled with a hapten, antigen or cofactor. The method of claim 1, wherein   54. The non-extendable nucleotide is dinitrophenol, lipoic acid or olefin. The method according to claim 1, wherein the method is labeled with a quinine compound.   55. The non-extendable nucleotide is labeled with a detectable polypeptide The method of claim 1, wherein   56. The non-extendable nucleotide is a molecule having a high electron density or an insoluble reaction product. The method of claim 1, wherein the method is labeled with an enzyme capable of depositing a product.   57. The non-extendable nucleotide is a molecule having a high electron density or an insoluble reaction product. The method of claim 1, wherein the method is labeled with an enzyme capable of depositing a product.   58. The fluorescent indicator molecule is fluorescein, rhodamine, Texas Red, FAM, JOE, TAMRA, ROX, HEX, TET, Cy3, Cy3.5, The group consisting of Cy5, Cy5.5, IRD40, IRD41 and BODIPY 49. The method of claim 48, wherein the method is selected from:   59. The electron-dense indicator molecule comprises ferritin, hemocyanin, and 58. The method of claim 57, wherein the method is selected from the group consisting of Lloyd gold.   60. The detectable polypeptide separates the detectable polypeptide from an indicator component. By specifically forming a complex with a second polypeptide covalently linked to the 56. The method of claim 55, which is detectable indirectly.   61. The detectable polypeptide is less than avidin and streptavidin. Selected from the group consisting of biotin and iminobi. 61. The method of claim 60, wherein the method is selected from the group consisting of otyne.   62. 17. The method of claim 16, wherein said nucleic acid molecule is from a plant.   63. 17. The method of claim 16, wherein said nucleic acid molecule is from a microorganism.   64. The microorganism is a bacterium, fungus, yeast, virus, viroid and other genetic 64. The method of claim 63, wherein the method is selected from the group consisting of possible genetic entities. .