WO2002046393A1 - Method of identifying nucleotide polymorphism - Google Patents

Abstract

It is intended to provide the following two methods: a method for identifying a nucleotide polymorphism contained in a nucleic acid sample which comprises treating a nucleic acid sequence containing a specific nucleotide polymorphism site in a sample simultaneously or separately with a wild type-specific primer and a polymorphism-specific primer, elongating these primers by DNA polymerases, and detecting the lengths of the elongation products of these primers or complementary strands thereof; and a method of simultaneously identifying two or more nucleotide polymorphisms contained in a nucleic acid sample which comprises treating a nucleic acid sequence containing two or more specific nucleotide polymorphism sites in a sample simultaneously or separately with two or more wild type-specific primers and two or more polymorphism-specific primers respectively corresponding to the nucleotide polymorphism sites, elongating these primers by DNA polymerases, and detecting the elongation products of these primers or complementary strands thereof.

Description

 Specification

 Nucleotide polymorphism identification method

 Technical field

 The present invention relates to a method for identifying a mutation or polymorphism in a nucleic acid sequence and a kit for detecting a base polymorphism. The present invention is particularly useful for diagnosis of genetic diseases, nucleotide polymorphism analysis, and the like.

 Background art

 In the present invention, the nucleotide polymorphism is a genotype having a sequence different from that of the wild-type, and the polymorphic gene plays an important role as a cause of inter-individual variation in the occurrence of side effects in drug metabolism and treatment failure. It is also known as a cause of individual differences such as basal metabolism known as constitution. In addition, they also serve as genetic markers for many diseases. Therefore, the elucidation of these mutations is clinically important, and phenotyping of rutin is particularly recommended for psychiatric patients and suicide volunteers in clinical research (Gram and Brsen, European Consensus Conference on Pharmacogenet i cs. Commi ss on of the European Communi ties, Luxembourg, 1990, pp. 87-96: Balant et al., Eur. J. Clin. Pharmacol. 554, (1989)). It is also desirable to have a nucleic acid sequence analysis method for detecting each polymorphism following the identification of the causative polymorphism gene.

 As a conventional nucleic acid sequence analysis technique, for example, there is a nucleic acid sequencing method (sequencing method). Nucleic acid sequencing can detect and identify nucleotide polymorphisms in nucleic acid sequences.However, preparation of type 铸 nucleic acids, DNA polymerase reaction, polyacrylamide gel electrophoresis, analysis of nucleic acid sequences, etc. It requires a great deal of labor and time to perform. In addition, although labor saving can be achieved by using a recent automatic sequencer, there is a problem that an expensive device is required.

 On the other hand, various genetic diseases caused by point mutations in genes are known, and among them, it is known which parts of genes and how point mutations cause genetic diseases. There are not many things.

As a method for detecting such an expected point mutation, a PCR (polymerase chain reaction) method (Japanese Patent Publication No. Hei 416-690, Japanese Patent Publication No. There is known a method for detecting a point mutation in a gene using a gene amplification method such as the method described in Japanese Patent Application Laid-Open No. 9-57. According to this method, one of a pair of primers used in the gene amplification method includes a normal primer completely complementary to an end region of an amplification region of a normal gene and an end portion of an amplification region of an abnormal gene as one primer. Use an aberrant primer completely complementary to the region. The abnormal primer has a nucleotide whose 3 'end is complementary to the nucleotide that caused the expected point mutation. The sample gene is subjected to a gene amplification method by using such normal and abnormal primers separately. If the sample gene is normal, nucleic acid amplification will occur when the normal primer is used, but when the abnormal primer is used, the 3 'end of the primer will be complementary to the corresponding nucleotide in the sample gene. Therefore, no elongation reaction occurs and no amplification of nucleic acid occurs. On the other hand, if the sample gene is abnormal, amplification does not occur when the normal primer is used, and amplification occurs when the abnormal primer is used. Therefore, by examining whether amplification occurs when each primer is used, it is possible to determine whether the sample gene is normal or abnormal, thereby detecting a point mutation in the sample gene. be able to. Alternatively, a nucleic acid sequence containing a point mutation may be amplified in advance, and a determination may be made as to whether an extension reaction occurs in each primer using the amplified nucleic acid. This method can also be applied to base polymorphism analysis. That is, polymorphism analysis becomes possible by using a wild-type specific primer in place of a normal type primer and a polymorphism specific primer in place of an abnormal type primer.

According to the above principle, it seems that the nucleotide polymorphism can be clearly detected by the conventional method, but actually, there is a difference of only one nucleotide between the wild-type specific primer and the polymorphic specific primer. When a wild-type gene is extended or amplified using a polymorphism-specific primer and when a polymorphism gene is extended or amplified using a wild-type specific primer, a certain degree of reaction often occurs. In many cases, it is difficult to make a clear judgment. Whether or not extension or amplification occurs when mismatched primers are used depends on the type of equipment used and other delicate conditions, and the reproducibility is low. Therefore, in order to completely prevent the reaction from occurring when a mismatched primer is used, it is necessary to control the temperature conditions during the reaction extremely strictly. Can be a difficult task.

 In addition, it is more difficult to use a DNA polymerase having 3 'exonuclease activity, which allows accurate extension reaction. In particular, if the 3'-end sequence is mismatched, the extension reaction may occur even though the extension of the mismatch is expected to occur by the polymerase proofreading function, despite the expectation that the extension reaction will not occur. There is a potential.

 On the other hand, various methods have been used to detect a nucleic acid fragment extended or amplified by the primer. One embodiment is a method of hybridization using a labeled oligonucleotide probe. This method is accurate because a signal is obtained only when the probe is hybridized, but the detection step becomes complicated and the detection becomes complicated. If many polymorphisms are measured at one time, other types of labels are required.

 An object of the present invention is to solve the above-mentioned problems and to provide a method capable of clearly and reproducibly detecting a polymorphism in a nucleic acid sequence and a reagent therefor. BRIEF DESCRIPTION OF THE FIGURES

 Figures 1 to 6 show the fluorescence measurement data obtained with the Applied Biosystems ABI PRISM 310 Genetic Analyzer. Disclosure of the invention

 In view of the above circumstances, the present inventors have conducted intensive studies and found that, in the above-described conventional method, when the wild-type specific primer hybridizes with the polymorphic nucleic acid, and when the polymorphic specific primer hybridizes with the wild-type nucleic acid, Measuring the length of the elongate allows easy and clear determination, and strict control of reaction conditions by associating a polymorphic nucleotide sequence with the second nucleotide from the 3 'end of the primer. The inventors have found that the primer extension reaction can be completely inhibited without performing this step, and have completed the present invention.

That is, the present invention relates to a nucleic acid sequence containing a specific nucleotide polymorphism site contained in a sample, A wild-type specific primer and a polymorphic specific primer, each having a different length or label, are allowed to act simultaneously or separately, the primer is extended with DNA polymerase, and the extension product of the primer and / or its complementary strand are extended. An object of the present invention is to provide a method for identifying a nucleotide polymorphism contained in a nucleic acid sample by detecting the length.

 Further, the present invention provides a nucleic acid sequence containing a specific nucleotide polymorphism site contained in a sample, together with a reverse primer, a wild-type specific primer and a polymorphism-specific primer having different lengths or labels, respectively, simultaneously or separately. Another object of the present invention is to provide a method for identifying a nucleotide polymorphism contained in a nucleic acid sample by detecting whether or not a specific nucleic acid fragment has been amplified.

The present invention relates to a nucleic acid sequence comprising 2 or more specific nucleotide polymorphism site contained in a sample, each nucleotide polymorphism length corresponding to the type site or 2 or more wild-type with different labeled specific primers and ₂ or more The primers simultaneously or separately, extend the primers with DNA polymerase, and detect an extension product of the primers and / or a complementary strand thereof to include the primers in the nucleic acid sample. It is intended to provide a method for simultaneously identifying the above nucleotide polymorphisms.

The present invention relates to a nucleic acid sequence comprising 2 or more specific nucleotide polymorphism site contained in a sample, each nucleotide polymorphism length corresponding to the type site or 2 or more wild-type with different labeled specific primers and ₂ or more The polymorphism-specific primers together with two or more reverse primers corresponding to each base polymorphism site, simultaneously or separately, extend the primers with DNA polymerase, and detect amplification products of the primers Thus, the present invention provides a method for simultaneously identifying two or more nucleotide polymorphisms contained in the nucleic acid sample.

 Further, the present invention provides a reagent kit for detecting a nucleotide polymorphism, wherein the second base from the 3 'end contains a primer, a DNA polymerase, a nucleotide and a size marker corresponding to a nucleotide expected to be polymorphic. Is provided.

In one preferred embodiment of the method for identifying a nucleotide polymorphism according to the present invention, the length of the wild-type specific primer and the length of the polymorphism-specific primer are different, whereby the length of the extension product is different. In another preferred embodiment of the method for identifying a nucleotide polymorphism according to the present invention, in the method for identifying a nucleotide polymorphism of a specific nucleic acid sequence contained in a sample, the second base from the 3 ′ end of the primer is a base. Use wild-type specific primers and one or more polymorphic specific primers corresponding to each predicted nucleotide at the polymorphic site.

 In another preferred embodiment of the method for identifying a nucleotide polymorphism according to the present invention, the wild-type specific primer and the polymorphism specific primer are not complementary to the specific nucleic acid sequence from the 3 'end to the 3' end from the 3 'end. Contains one nucleotide.

 In another preferred embodiment of the method for identifying a nucleotide polymorphism according to the present invention, the method for detecting length is any one of electrophoresis, mass spectrometry, and liquid chromatography.

 In another preferred embodiment of the method for identifying a nucleotide polymorphism according to the present invention, the DNA polymerase used in the primer extension reaction has 3 ′ exonuclease activity of double-stranded DNA.

 In another preferred embodiment of the method for identifying a nucleotide polymorphism according to the present invention, the DNA polymerase is Pyrococcus sp. i 1 ic archaebacterium).

 In another preferred embodiment of the method for identifying a nucleotide polymorphism according to the present invention, in the method for identifying a nucleotide polymorphism of a specific nucleic acid sequence contained in a sample, the nucleic acid sequence is amplified in advance.

 In another preferred embodiment of the method for identifying a nucleotide polymorphism according to the present invention, the nucleic acid amplification method is any one selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA.

 In another preferred embodiment of the method for identifying a nucleotide polymorphism according to the present invention, the wild-type-specific primer and the polymorphism-specific primer are pre-labeled.

 In another preferred embodiment of the method for identifying a nucleotide polymorphism of the present invention, the label is any one selected from the group consisting of an enzyme, a biotin, a fluorescent substance, a hapten, an antigen, an antibody, a radioactive substance, and a luminophore. is there.

Hereinafter, the present invention will be described in detail. When a nucleic acid sequence is replicated, a primer, four types of deoxynucleoside triphosphates (dNTPs), and a DNA polymerase are allowed to act on a single-stranded denatured target nucleic acid, and the primer extension reaction is performed using the target nucleic acid as a 铸 -type. The complementary strand of the resulting nucleic acid sequence is synthesized. At this time, the polymorphism can be detected by performing an extension reaction using the wild-type specific primer and the polymorphism specific primer separately for the sample nucleic acid.

 At this time, if the target nucleic acid is not contained in an amount sufficient for detection, it is possible to amplify the nucleic acid fragment containing the polymorphic sequence in advance. Examples of the nucleic acid amplification method in this case include PCR, NASBA (Nucleic acid sequence-based amplification method; Nature, Vol. 350, p. 91 (1991)), LCR (International Publication No. WO 89/12696, Kaihei 2_29344), SDA (Strand Displacement Amplification: Nucleic acid reserch Vol. 20, p. 1691 (1992)), RCR (International Publication No. 90/1069), TMA (Transcript ion mediated amplification method; J. Clin. Microbiol. Vol. 31, p. 3270 (1993)). In particular, the PCR method involves repeating a cycle consisting of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of primers, and a thermostable DNA polymerase. This is a method of exponentially amplifying a sample nucleic acid region sandwiched between the pair of primers. That is, the nucleic acid of the sample is denatured in the denaturation step, each primer is hybridized with a region on the single-stranded sample nucleic acid complementary to each other in the subsequent annealing step, and in the subsequent elongation step, the DNA is generated starting from each primer. The DNA chain complementary to each single-stranded sample nucleic acid that becomes a type II by the action of the polymerase is extended to form a double-stranded DNA. By this one cycle, one double-stranded DNA is amplified to two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of primers is theoretically amplified 2 n times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, using the gene amplification method, it is possible to detect a very small amount (even a single molecule) of sample nucleic acid, which was previously undetectable, and it is a very widely used technology in recent years. .

In the method using the nucleic acid amplification method, a wild type specific that can amplify a wild type nucleic acid: The gene amplification method can be carried out by separately using polymorphism-specific primers capable of amplifying the polymorphic nucleic acid. Here, a polymorphic nucleic acid is a wild-type nucleic acid in which only one nucleotide of the wild-type nucleic acid is point-mutated and replaced with another nucleotide, or inserted or deleted in a part of the wild-type nucleic acid sequence. Nucleic acid sequence containing, and it is elucidated at which site the nucleotide is mutated. It has been elucidated that the constitutions and the like differ depending on such nucleotide polymorphisms. The method of the present invention is a method for examining whether or not a sample nucleic acid has such expected polymorphism. is there.

 When a sample nucleic acid is extended or amplified using a wild-type specific primer, the reaction occurs when the sample nucleic acid is wild-type, but does not occur when the polymorphism is used. Conversely, when a sample nucleic acid is extended or amplified using polymorphism-specific primers, the reaction occurs if the sample nucleic acid is polymorphic, but does not occur if the sample nucleic acid is wild-type. Therefore, one sample was divided into two, one was reacted with the wild-type specific primer, and the other was reacted with the polymorphic specific primer, to determine whether the reaction had occurred. It is possible to clearly determine whether the sample nucleic acid is wild-type or polymorphic. In particular, higher organisms, including humans, have one father-derived gene and one maternal-derived gene for one type of gene. You can also distinguish between homozygous and polymorphic or heterozygous. That is, in the case of heterozygous reaction, a wild-type gene and a polymorphic gene are both present, so that a reaction occurs both when a wild-type specific primer is used and when a polymorphic specific primer is used.

 Although what has been described so far is common to the conventional method, as described above, in the conventional method, the combination of the wild-type specific primer-Z polymorphic nucleic acid and the polymorphic specific primer 'Since only the terminus is non-complementary (mismatch) to type III nucleic acid (ie, the 3' terminus of the primer corresponds to the expected polymorphic site in the sample nucleic acid), the reaction also occurs in these combinations There are cases. In such a case, it is difficult to determine whether the sample nucleic acid is wild type or polymorphic.

This is because DNA polymerase derived from Thermus aquaticus, which is often used in amplification reactions, does not have 3 'exonuclease activity. When the complementary strand of the sequence could not be synthesized Since the amplification reaction is continued as it is, the amplified nucleic acid fragment may contain a mutation. In other words, even if there is a mismatch at the 3 'end, the reaction proceeds, and it is thought that such a problem occurs.

 On the other hand, DNA polymerase derived from Pyrococcus sp. K0D1 strain or Hyperthermophilic archaebacterium, which has excellent extension reaction accuracy, If there is a mismatch at the 3 'end due to its 3' exonuclease activity, this problem may occur because the nuclease activity removes the mismatch and continues the extension reaction.

In order to solve such a problem, in one of the preferred embodiments of the present invention, the arrangement of the primers is devised. That is, the primer used in the present invention is designed such that the second nucleotide from the 3 ′ end of the primer in the above combination corresponds to the nucleotide of the polymorphic sequence. In the case of such a design, in the combination of wild-type specific primers, wild-type nucleic acid and polymorphism-specific primer Z polymorphism nucleic acid, the primers are completely identical, so that the reaction occurs. In the combination of type-specific primer Z wild-type nucleic acid, since the second base from the 3 'end of the primer is mismatched, DNA polymerase having 3' exonuclease activity recognizes the mismatch. 'It was confirmed that the exonuclease did not work because the ends were complementary, and that no DNA polymerase reaction occurred. Thus, by detecting a polymorphism-specific extension reaction product or amplification product, a nucleotide polymorphism in a specific sequence can be identified. As a conventional nucleic acid sequence detection technique, there is, for example, the Southern hybridization method (Masami Muramatsu, “Lab Manual Genetic Engineering Supplement”, published by Maruzen Co., Ltd., pp. 70-75). According to this method, a region of DNA having a base sequence complementary to the labeled DNA probe can be identified. That is, in the Southern hybridization method, nucleic acid fragments are electrophoresed on an agarose gel-polyacrylamide gel plate, separated according to the size (length) of the fragments, and denatured into single strands. Then, attach a membrane such as nitrocellulose or nylon to the plate, and transfer the nucleic acid fragments as they are in the electrophoresis pattern. After immobilization, a hybrid is formed with a nucleotide polymorphism-specific DNA probe labeled with RI (radioisotope) or the like, and the nucleic acid fragment on the membrane complementary to the probe is detected by autoradiography or the like.

According to the Southern hybridization method, the electrophoretic position and molecular weight of the target DNA fragment can be determined.In the present invention, however, only the presence or absence of amplification is used for the gene polymorphism because the above-described specific primers are used. Can identify the polymorphism. Electrophoresis is the simplest method. That is, after performing an extension reaction using a specific primer, it is possible to identify the polymorphism by confirming the length of the extension product or amplification product by electrophoresis. At this time, if the length of the extension or amplification product differs between the wild-type specific primer and the polymorphic specific primer, the polymorphism can be easily identified by electrophoresis, particularly by a genetic analyzer. Furthermore, when the length of the extension or amplification product differs depending on the type of polymorphism, it is possible to identify many types of polymorphisms at once. In addition, it is also possible to use a method for identifying the molecular weight, such as mass spectrometry or liquid chromatography. A sequencer for determining a nucleic acid sequence can accurately detect a difference in the length of one base by electrophoresis. In the present invention, by utilizing the features of the sequencer, the length of an extension product or an amplification product is adjusted using a wild-type specific primer and one or two polymorphism-specific primers corresponding to the number of polymorphisms. In this way, multiple types of polymorphisms can be accurately identified in one electrophoresis. Therefore, even when there are two or more polymorphic sites, the number of nucleotides of at least one wild-type specific primer and at least one polymorphic specific primer to be used simultaneously and the length of the extension product or the reverse primer are amplified. If the lengths of the amplification products are designed to be different from each other, even if they are used simultaneously, the lengths of the amplification products and extension products are different, so that it is easy to detect which primer amplification product was obtained. be able to. In the present invention, as a primer for detecting a polymorphism, a primer set consisting of a wild-type specific primer, at least one polymorphism-specific primer and a reverse primer can be preferably used. Since the number of bases of the extended or amplified nucleic acid is determined by one of the wild-type or polymorphism-specific primers and the reverse primer, the wild-type-specific primer and at least one polymorphism-specific primer In the first case, those having different numbers of bases are used. The number of polymorphism-specific primers is the same as the number of polymorphism bases. For example, if the wild-type base is G and the polymorphism bases A and C are known, the type A polymorphism-specific primer If two types of primers and C-type primer are used, and if the polymorphic base is only A, use one (A-type) polymorphism-specific primer. The chromosome containing a specific nucleotide polymorphism site or a fragment thereof contained in a sample is not particularly limited as long as it is a target nucleic acid containing a nucleotide polymorphism site that carries information on a target gene. Examples of the target nucleic acid include an Alu sequence, exons and introns of a gene encoding a protein, and a promoter. More specifically, it includes genes related to various diseases including genetic diseases, drug metabolism, and lifestyle-related diseases (such as hypertension and diabetes). For example, hypertension includes the ACE gene.

 In the present invention, a polymorphic nucleic acid of a chromosome or a nucleic acid fragment to be measured for a nucleotide polymorphism is a wild-type nucleic acid in which at least one nucleotide is point-mutated and replaced with another nucleotide, It is a nucleic acid containing an inserted or deleted sequence as part of a wild-type nucleic acid. It has been elucidated that the constitutions and the like differ depending on such nucleotide polymorphisms, and the method of the present invention is a method for examining whether or not a nucleic acid in a sample has such an expected polymorphism. It is.

 The length of each oligonucleotide primer in the present invention is 13 to 35 bases, preferably 16 to 30 bases.

 In the present invention, the kit includes a primer (wild-type-specific primer and one or two or more polymorphism-specific primers) in which the second base from the 3 ′ end corresponds to a nucleotide predicted to be polymorphic, a reverse primer, DNA polymerase, containing nucleotides (dATP, dGTP, dTTP and dCTP).

 When two or more nucleotide polymorphisms are simultaneously detected, even if one kit contains the number of wild-type specific primers corresponding to the number of nucleotide polymorphisms and at least one polymorphism-specific primer Good. Two or more nucleotide polymorphisms are closely related to one disease, constitution, and the like, and a group of nucleotide polymorphisms that can determine the risk of a disease by detecting them at the same time is exemplified.

When two or more nucleotide polymorphisms are detected simultaneously, the same number of wild-type The total number of polymorphism-specific primers corresponding to the primers and each polymorphism site will be used. In this case, any combination of the wild-type specific primer and the polymorphic specific primer may be used.

 As described above, according to the method of the present invention, many polymorphisms can be simultaneously identified without generating false positives as in the conventional method. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Example 1

 Detection of nucleotide polymorphisms in the MTH F R (5, 10—Methylenetetrahydrofolate reducase) gene

 (1) Primer for detecting polymorphism in MTH F R gene

 A 19-base oligonucleotide having the base sequence of SEQ ID NO: 1 (hereinafter, referred to as primer 1), an oligonucleotide having the base sequence of SEQ ID NO: 2 (hereinafter, referred to as primer 1-2), and SEQ ID NO: 3 An oligonucleotide having the nucleotide sequence shown in (1) (hereinafter, referred to as Primer 13) was obtained by requesting Japan Bioservices to synthesize.

 Primer 1 has the nucleotide sequence at the second base from the 3 'end as a wild-type (C) nucleotide, has a 5' end labeled with 6 FAM, and primer 1-2 has the second base from the 3 'end. It has a nucleotide sequence of polymorphism (T), is labeled at the 5 'end with TAMRA, and primer 3 is a reverse primer paired with both primers 1 and 2.

The primer 1 has 19 bases and the primer 1 has 21 bases, and the primers 1 and 2 were designed so as to be able to detect the difference in the length of these 2 bases. In order to detect the difference in length, the difference in the number of base pairs between the two primers may be one or more, preferably one to three. (2) Analysis of MTHFR gene polymorphism

 Using a DNA solution extracted from human leukocytes by the phenol-chloroform method as a sample, the following reagents were added, and the human MTHFR gene polymorphism was analyzed under the following conditions.

 (a) Reagent

 A 25 PL 1 solution containing the following reagents was prepared.

 Primer 1 and 2 5 pmol each

 Primer 3 5 pmo 1

 X 10 buffer 2.5 1

 2mM dNTP 2.5 1

25mM Mg S0 ₄ 1 1

 KODplus DNA polymerase 0.2 U

 Extracted DNA solution l O Ong

Amplification conditions

 942 minutes

 94 '15 seconds, 57.5' 30 seconds, 68 ^ · 30 seconds (35 cycles) 68 · 2 minutes

 (b) Detection

 The obtained PCR product was diluted 10-fold, and the 11 was diluted with 19 1 Te immediate late Suppression Reagent (Applied Biosystems), followed by ice-cooling after 942 min of reaction. After centrifugation, the amplified product was detected using a Genetic Analyzer ABI PRISM310 from Applied Biosystems. As a result, the results shown in Figs. 1 to 3 below were obtained.

(3) Explanation of figure

Figure 1

An amplification product was detected using a sample in which the polymorphism of the MTHFR gene was CZC. As a result, a peak of only C type labeled with 6 FAM was observed at 61 bp. Figure 2

 An amplification product was detected using a sample in which the polymorphism of the MTHFR gene was CZT. As a result, two peaks of 6 FAM-labeled C type at 61 bp and TAMRA-labeled T type at 63 bp were observed.

Fig. 3

 An amplification product was detected using a sample in which the polymorphism of the MTHFR gene was TZT. As a result, a peak of only TAMRA-labeled T type was observed at 63 bp.

 As described above, the genotype could be clearly determined by detecting the color and length of the label of the amplification product using a primer containing a polymorphic sequence at the second base from the 3 ′ end. Example 2 Simultaneous Detection of Nucleotide Polymorphism of MTHFR (5, 10— Methylenetetrahydrofolate reductase) Gene and 2C19 Gene Polymorphism of Cytochrome P450

 (1) primers for detecting polymorphisms in the MTHFR gene

 Primers 1, 2, and 3 used in Example 1 were used.

 (2) Primer for detecting polymorphism of 2C19 gene

 A 24-base oligonucleotide having the base sequence shown in SEQ ID NO: 4 (hereinafter referred to as primer 4), a 25-base oligonucleotide having the base sequence shown in SEQ ID NO: 5 (hereinafter referred to as primer 5), and SEQ ID NO: An oligonucleotide having the nucleotide sequence shown in No. 6 (hereinafter, referred to as primer 6) was obtained by requesting Nippon Bioservice to synthesize it.

 Primer 4 has a wild-type (G) nucleotide sequence at the second base from the 3 ′ end, has a 5 ′ end labeled with 6 FAM, and primer 5 has a base at the second base from the 3 ′ end. It has the nucleotide sequence of polymorphism (A), is labeled at the 5 'end with TAMRA, and primer 16 is a reverse primer paired with both primers 4 and 5.

(3) Simultaneous analysis of MTHFR gene polymorphism and 2C19 gene polymorphism Using a DNA solution extracted from human leukocytes by the phenol-chloroform method as a sample, the following reagents were added, and analyzed under the following conditions.

(a) Reagent

 A 25 1 solution containing the following reagents was prepared.

 Primer 1 and 2 5 pmo 1 each

 Primer 3 5 pmo 1

 Primers 4 and 5 5 pmo 1 each

 Primer 6 5 pmo 1

 X 10 buffer 2.5 1

 2mM dNTP 2.5 1

25mM Mg S0 ₄ 1 1

 KODplus DNA polymerase 0.2 U

 Extracted DNA solution 100 ng

Amplification conditions

 942 minutes

 94 15 seconds, 57.5 · 30 seconds, 68 ° C · 30 seconds (40 cycles) 68 · 2 minutes

 (b) Detection

 The resulting PCR product was diluted 10-fold, and the 11 was diluted with 91 1 of Template Suppression Reagent (Applied Biosystems), and cooled with ice after 942 min of reaction. After centrifugation, the amplified products were detected with the Applied Biosystems Genetic Analyzer ABI PRISM310. As a result, the results shown in Figs. 4 to 6 below were obtained.

(4) Explanation of figure

Fig. 4

Amplification products were detected using a sample in which the polymorphism of the MTHFR gene was C / C and the genotype of 2C19 was GZG. As a result, 6 to 8 ^ 1 markers were assigned to 61 of the MTHFR gene. And a G-type peak labeled with 6 FAM at 77 bp of the 2C19 gene.

Fig 5

 Amplification products were detected using a sample in which the polymorphism of the MTHFR gene was CZT and the genotype of 2C19 was GZA. As a result, the 6FAM-labeled C-type peak at 61 bp of the MTHFR gene and the TAMRA-labeled T-type peak at 63 bp, and the 6FAM-labeled G-type peak and 78 bp at 77 bp of the 2C19 gene at 63 bp. Four of the type A peaks of TAMR A label were observed.

Fig. 6

 Amplification products were detected using a sample in which the polymorphism of the MTHFR gene was TZT and the genotype of 2C19 was AZA. As a result, two peaks, a T-type TAMRA-labeled peak at 63 bp of the MTHFR gene and a TAMRA-labeled A-type peak at 78 bp of the 2C19 gene were observed.

 As described above, two types of gene polymorphisms should be clearly determined by detecting the color and length of the label of the amplification product using a primer containing a polymorphic sequence at the second base from the 3 'end. Was completed.

 Example 2 is an example in which two types of gene polymorphisms are simultaneously detected. Similarly, by simultaneously using three or more types of primer sets (for example, primers 1, 2, and 3 constitute one primer set) Similarly, three or more nucleotide polymorphisms can be detected. However, in this case, if the number of bases of the amplification product detected by each primer set (for example, 61 bp and 63 bp when a primer set consisting of primers 1 to 3 is used) is different, the number of bases of the amplification product It is possible to detect nucleotide polymorphisms based on the difference in length. For example, when a primer set consisting of primers 1 to 3 is used, the number of bases of the 61 bp (C type) amplification product is determined by primer 1 and primer-3, so the complementary position of reverse primer 3 may be shifted. Alternatively, by increasing or decreasing the length of the primer 11, the number of bases of the amplification product can be easily adjusted.

As described above, according to the present invention, polymorphism in a sample nucleic acid can be clearly and easily detected. A method is provided. In the method of the present invention, since false positives do not occur, the results can be obtained with good reproducibility even if the conditions of the gene amplification method are not so strict, and the judgment results do not differ depending on the type of the model. Further, according to the method of the present invention, it has become possible to identify many types of polymorphisms at one time.

Claims

The scope of the claims  1. A wild-type specific primer and a polymorphism specific primer are allowed to act simultaneously or separately on a nucleic acid sequence containing a specific nucleotide polymorphism site contained in a sample, and the primer is extended with DNA polymerase, A method for identifying a base polymorphism contained in a nucleic acid sample by detecting the length of a primer extension product and / or its complementary strand.  2. The method according to claim 1, wherein the length of the wild-type specific primer and the length of the polymorphic specific primer are different, whereby the length of the extension product is different.  3. In the method for identifying a nucleotide polymorphism in a specific nucleic acid sequence contained in a sample, a wild-type specific primer in which the second base from the 3 ′ end of the primer corresponds to each nucleotide at the nucleotide polymorphism site expected The method according to claim 1, wherein one and one or more polymorphism-specific primers are used.  4. The method according to claim 3, wherein the wild-type specific primer and the polymorphic specific primer contain one nucleotide that is not complementary to the specific nucleic acid sequence from the 3 'end to the 3' to 5 'end.  5. The method according to claim 1, wherein the method for detecting the length is any one of electrophoresis, mass spectrometry, and liquid chromatography.  6. The method according to claim 1, wherein the DNA polymerase used in the primer extension reaction has 3 ′ exonuclease activity of double-stranded DNA.  7. The method according to claim 6, wherein the DNA polymerase is derived from Pyrococcus sp. K0D1 strain or Hyperthermophilic archaebacterium.  8. The method according to claim 1, wherein in the method for identifying a nucleotide polymorphism of a specific nucleic acid sequence contained in a sample, the nucleic acid sequence is amplified in advance.  9. The method according to claim 8, wherein the nucleic acid amplification method is any one selected from the group consisting of FCR, NASBA, LCR, SDA, RCR and TMA.  10. The method according to claim 1, wherein the wild-type specific primer and the polymorphic specific primer are labeled in advance. 11. Label the enzyme, biotin, fluorescent substance, hapten, antigen, antibody, radioactive substance, 11. The method according to claim 10, which is any one selected from the group consisting of:  12. A wild-type specific primer and a polymorphic specific primer are simultaneously or separately acted on a nucleic acid sequence containing a specific nucleotide polymorphism site contained in a sample together with a reverse primer to amplify the nucleic acid sequence. A method for identifying nucleotide polymorphisms contained in a nucleic acid sample by detecting the length of the amplification product to determine whether or not the DNA has been amplified.  13. The method according to claim 12, wherein the length of the wild-type specific primer and the length of the polymorphic specific primer are different, whereby the length of the extension product is different.  14. In a method for identifying a nucleotide polymorphism in a specific nucleic acid sequence contained in a sample, a wild-type specific primer in which the second base from the 3 'end of the primer corresponds to each nucleotide at the nucleotide polymorphism site expected 13. The method according to claim 12, wherein one and one or more polymorphism-specific primers are used.  15. The method according to claim 14, wherein the wild-type specific primer and the polymorphic specific primer contain one nucleotide that is not complementary to the specific nucleic acid sequence from the 3 'end to the 3' to 5 'end.  16. The method according to claim 12, wherein the method for detecting the length is any one of electrophoresis, mass spectrometry, and liquid chromatography.  17. The method according to claim 12, wherein the DNA polymerase used in the primer extension reaction has 3 ′ exonuclease activity of double-stranded DNA.  18. The method according to claim 12, wherein the DNA polymerase is derived from Pyrococcus sp. K0D1 strain or Hyper thermophilic archaebacterium.  19. The method according to claim 1, wherein the second base from the 3 ′ end contains a primer corresponding to a nucleotide predicted to be polymorphic, a DNA polymerase, a nucleotide, and a size marker. Reagent kit for type detection. 20. Simultaneously add two or more wild-type specific primers and two or more polymorphism-specific primers corresponding to each nucleotide polymorphism site to the nucleic acid sequence containing two or more specific nucleotide polymorphism sites contained in the sample. Alternatively, the primers are extended with a DNA polymerase, and the extension product of the primer and / or its complementary strand are detected to detect its nucleus. A method for simultaneously identifying two or more nucleotide polymorphisms contained in an acid sample.  21. The method according to claim 20, wherein the lengths of the wild-type specific primer and the polymorphic specific primer are different.  22. In the method for identifying nucleotide polymorphisms in two or more specific nucleic acid sequences contained in a sample, the second base from the 3 'end of the primer corresponds to each nucleotide at the predicted nucleotide polymorphism site 21. The method according to claim 20, wherein a wild-type specific primer and one or more polymorphic specific primers are used.  23. The method according to claim 22, wherein the wild-type specific primer and the polymorphic specific primer contain nucleotides not complementary to the specific nucleic acid sequence from the 3 'end to the 3' end to the 5 'end.  24. The method according to claim 20, wherein the method for detecting the length is any one of electrophoresis, mass spectrometry, and liquid chromatography.  25. The method according to claim 20, wherein the DNA polymerase used in the primer extension reaction has 3, exonuclease activity of double-stranded DNA.  26. The method according to claim 20, wherein the DNA polymerase is derived from Pyrococcus sp. K0D1 strain or Hyperthermophilic archaebacterium.  27. The method according to claim 20, wherein in the method for identifying a nucleotide polymorphism of a specific nucleic acid sequence contained in a sample, the nucleic acid sequence is amplified in advance.  28. The method according to claim 27, wherein the nucleic acid amplification method is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCR and TMA.  29. The method according to claim 20, wherein the wild-type specific primer and the polymorphic specific primer are labeled in advance.  30. The method according to claim 29, wherein the label is any one selected from the group consisting of an enzyme, a biotin, a fluorescent substance, a hapten, an antigen, an antibody, a radioactive substance, and a luminophore. 31. Two or more wild-type specific primers and two or more polymorphic specific primers corresponding to each nucleotide polymorphism site are added to the nucleic acid sequence containing two or more specific nucleotide polymorphism sites contained in the sample, respectively. With two or more reverse primers corresponding to the nucleotide polymorphism site Simultaneously or separately, amplifying with a DNA polymerase, and simultaneously detecting two or more nucleotide polymorphisms contained in the nucleic acid sample by detecting the amplification product of the primer.  32. The method of claim 31, wherein the wild-type specific primer and the polymorphic specific primer have different lengths, thereby resulting in different extension products. 33. In the method for identifying nucleotide polymorphisms in a specific nucleic acid sequence contained in a sample, the second base from the 3 'end of the primer has a wild-type specificity corresponding to each nucleotide predicted at the nucleotide polymorphism site. The method according to claim 31, wherein a primer and one or more polymorphism-specific primers are used.  34. The method according to claim 33, wherein the wild-type specific primer and the polymorphic specific primer contain one nucleotide that is not complementary to a specific nucleic acid sequence from the 3 'end to the 3' end.  35. The method according to claim 31, wherein the method for detecting the length is any one of electrophoresis, mass spectrometry, and liquid chromatography.  36. The method according to claim 31, wherein the DNA polymerase used in the primer extension reaction has 3 'exonuclease activity of double-stranded DNA. 37. The method according to claim 36, wherein the DNA polymerase is derived from Pyrococcus sp. K0D1 strain or Hyperthermophilic archaebacterium.  38. The method according to claim 20 to claim 37, wherein the second base from the 3 'end contains a primer, a DNA polymerase, a nucleotide, and a size marker corresponding to the predicted nucleotide of the polymorphism. Characteristic reagent kit for detecting base polymorphisms.