WO2006070666A1

WO2006070666A1 - Method of simultaneously detecting gene polymorphisms

Info

Publication number: WO2006070666A1
Application number: PCT/JP2005/023485
Authority: WO
Inventors: Masamitsu Shimada; Fumitsugu Hino; Ikunoshin Kato; Kazuyuki Kawakami
Original assignee: Takara Bio Inc.
Priority date: 2004-12-28
Filing date: 2005-12-21
Publication date: 2006-07-06
Also published as: JPWO2006070666A1

Abstract

A method of detecting polymorphisms characterized by comprising simultaneously detecting a variable number of tandem repeats (VNTR) in a target nucleic acid and a single nucleotide polymorphism (SNP) occurring in the repeat unit sequence, which involves the step of simultaneously hybridizing the target nucleic acid having the polymorphisms to be detected with at least three oligonucleotides; and a composition and a kit to be used in the above method.

Description

Simultaneous detection method of gene polymorphism

Technical field

[0001] The present invention relates to a method, composition, and kit for rapidly and conveniently detecting a VNTR of a gene related to disease prediction, drug efficacy and side effects and SNPs present in the repeat unit sequence of the VNTR with high sensitivity.

Background art

[0002] It is known that there is a difference in base sequence called polymorphism that is not the same in the genetic code contained in the genome of an individual organism belonging to the same species. Known polymorphisms include deletions and insertions of 1 to several tens of bases, and those with specific base sequence duplication (repetitive sequence polymorphism: V NTR), but one base is replaced by another base. Those that are substituted are called single nucleotide polymorphisms (SNPs).

[0003] As a method for detecting VNTR, the method of directly decoding the base sequence, the restriction fragment length polymorphism (RFLP) analysis method, the nucleic acid amplification reaction, and then determining the amplified fragment length by agarose gel electrophoresis The method to do is used.

[0004] Methods for detecting SNPs are broadly classified into those based on hybridization, those based on primer extension, and those utilizing the substrate specificity of an enzyme.

For example, cycling probe reaction (for example, Patent Document 1), TaqMan method (for example, Patent Document 2), primer that anneals the 3 'end to the base part where DNA substitution is to be detected. A method for detecting the presence or absence of a primer extension reaction using a primer (for example, Patent Document 3), using a primer in which the base substitution site to be detected is located at the second nucleotide from the 3 'end Whether or not there is an extension reaction Method of detecting base substitution (for example, Patent Document 4), using a primer whose 3 'end is annealed to the base adjacent to the 3' side of the base to be detected for base substitution. , A method for determining the presence or absence of a mutation in the target part by determining the base incorporated into the primer, a method using a DNA ligase, an enzyme Chromatography _(Invader) method (for example, Patent Document 5) have been developed, Ru. [0005] Thymidylate Synthase (TS) is an enzyme that synthesizes 5 'thymidylate (dTMP) from 5' deoxyuridylate (dUMP) in cells. The gene sequence of human TS (hTS) It has been shown that there is a 28 bp triple repeat in the 5 'UTR region (eg, Non-Patent Document 1).

[0006] Furthermore, it has been shown that the 28 bp repeat of the 5 'UTR region of the TS gene forms a repeat polymorphism (VNTR) with 2 repeats (2R) and 3 repeats (3R). (For example, Non-Patent Document 2) Next, the 3R sequence of 5 'UTR clearly shows that TS expression is enhanced, and a pile cancer agent that targets TS is 5-FU (5-fluorouracil) treatment. An association was suggested (eg, non-patent literature 3). Based on the above findings, methods for detecting VNTR present in the 5 ′ UTR region of the TS gene have been reported (for example, Patent Document 6 and Patent Document 7). Patent Document 7 discloses a method capable of detecting only VNTR by hybridization of 3R-specific oligonucleotide hybrids, which is detection based on the possibility of hybridization based on a mismatch of power bases. Therefore, detection under the conditions of ibridiz is required.

[0007] In addition to the above VNTR, it has been clarified that the polymorphism in the 5 'UTR region of the TS gene has a single nucleotide substitution polymorphism (SNP) in the repetitive sequence of the VNTR (for example, a patent Reference 8). In Patent Document 1, the 5 'UTR region of the TS gene is amplified by PCR, half of the amplified product is subjected to agarose gel electrophoresis, VNTR is determined by the length of the amplified product, and the remaining half is restricted. A method for detecting SNPs by fragment length polymorphism (RFLP) analysis technology is disclosed. The relationship between the above SNP and the efficacy of 5-FU has been reported (for example, Non-Patent Document 4 and Non-Patent Document 5). Furthermore, the 3RG allele is evaluated as a polymorphism with high TS expression, and it has been shown that it can be used to determine the efficacy and side effects of 5FU (eg, Non-Patent Document 6). In particular, only the 2RZ3R gene polymorphism in the Orientals was not associated with 5-FU drug efficacy and side effects, indicating that the 3RG polymorphism has a significant effect (eg, non-patent literature). 7).

[0008] The conventional SNP analysis method is difficult to apply to the SNP present in the repeat unit sequence of VNTR because SNP and its surrounding sequences are repeated, and VNTR cannot be detected. Therefore, analysis of SNPs currently present in the VNTR and the repetitive unit sequence of the VNTR is performed by amplifying the target gene by PCR and dividing the amplified product into multiple tubes. The amplification product is purified as necessary, digested with several restriction enzymes, and RFLP analysis is performed by gel electrophoresis. However, the above method is time consuming (24 hours), the result determination is complicated, and a complicated inspection operation is required. In epidemiological studies and large-scale clinical trials, many problems have arisen, such as cross-contamination resulting in incorrect typing results by opening tubes after PCR reaction.

Patent Document 1: U.S. Patent No. 5,660, 988

Patent Document 2: U.S. Pat.No. 5,210,015

Patent Document 3: U.S. Pat.No. 5,137,806

Patent Document 4: International Publication No.2001Z42498 Pamphlet

Patent Document 5: U.S. Pat.No. 5,846,717

Patent Document 6: International Publication No.2001Z36686 Pamphlet

Patent Document 7: International Publication No. 2004Z031408 Pamphlet

Patent Document 8: International Publication No. 2004Z037852 Pamphlet

Non-patent literature l: Takeishi K, 5 others, Nucleic Acids Res. 1985, vol. 13, p. 2035-43

Non-Patent Document 2: Horie N, 4 others, Cell Structure Function, 1995, vol. 20, p. 191-7

Non-Patent Document 3: Kawakami K and 3 others, Anticancer Res. 1999, vol. 19, p. 3 249-52

Non-Patent Document 4: Mandola M V, 6 others, Cancer Res. 2003, vol. 63, p. 289 8-2904

Non-Patent Document 5: Kawakami K, 1 other, Cancer Res. 2003, vol. 63, p. 6004

-7

Non-Patent Document 6: Marcuello E, 5 others, Int. J. Cancer 2004, vol. 112, p. 73 3-737

Non-Patent Document 7: Kazuyuki Kawakami, Bio Industry, 2004, vol. 21, p. 50-57 Invention Disclosure

Problems to be solved by the invention [0010] The object of the present invention has been made in view of the above-described prior art, and is a method for simultaneously and simply detecting with high sensitivity, the typing of SNPs present in the repeating unit sequence of VNTR and VNTR. And providing a composition and a kit for use in the method.

Means for solving the problem

[0011] The inventors of the present invention have a method for detecting VNTR and SNP typing present in the repeating unit sequence of VNTR at the same time, simply and with high sensitivity, by simultaneously hybridizing at least three oligonucleotides. Compositions and kits used in the method were constructed to complete the present invention.

[0012] That is, in the first invention of the present invention, detection of a repetitive sequence polymorphism (VNTR) in a target nucleic acid and detection of a single nucleotide polymorphism (SNP) present in the repetitive unit sequence of the VNTR are performed simultaneously. The present invention relates to a polymorphism detection method characterized in that the method comprises a step of simultaneously hybridizing a target nucleic acid for detecting a polymorphism and at least three oligonucleotides. In the first invention of the present invention, three oligonucleotides are:

(1) a labeled oligonucleotide that identifies SNPs present in the repeat unit sequence of VNTR,

(2) an oligonucleotide that hybridizes to the boundary region between the most upstream repetitive sequence in the repetitive sequence and the upstream sequence adjacent to the repetitive sequence;

(3) An oligonucleotide that hybridizes to a boundary region between a repetitive sequence that is the most downstream among the repetitive sequences and a downstream sequence adjacent to the repetitive sequence. Furthermore, hybridization may be performed simultaneously with the amplification reaction of the target nucleic acid for detecting the polymorphism.

[0013] A second invention of the present invention includes the following steps:

(1) A labeled oligonucleotide that identifies an SNP present in a repeat unit sequence of VNTR, and an oligonucleotide that hybridizes to the boundary region between the most upstream repeat sequence and the upstream sequence adjacent to the repeat sequence 3 oligonucleotides consisting of oligonucleotides that hybridize to the border region between the most downstream repeat sequence and the downstream sequence adjacent to the repeat sequence, and the target nucleic acid to detect polymorphisms Preparing a reaction solution containing a sample suspected of having a primer, a primer for amplifying a target nucleic acid for detecting a polymorphism;

(2) Reaction that amplifies target nucleic acid to detect polymorphism in reaction solution prepared in step (1) Subjecting to; and

(3) the process of amplification reaction and detecting the signal derived from Z or labeled oligonucleotide in the reaction solution after the reaction;

The present invention relates to a method for detecting a polymorphism, comprising simultaneously detecting VNTR in a target nucleic acid including, and detecting SNP present in the repeating unit sequence of the VNTR.

[0014] In the first and second inventions of the present invention, the target nucleic acid for detecting the polymorphism may be a nucleic acid corresponding to the 5 'untranslated region of the thymidylate synthase (TS) gene. In this case, the group consisting of the oligonucleotides described in SEQ ID NOs: 1 and 2 in the sequence listing, the group consisting of the oligonucleotides described in SEQ ID NOS: 3 and 12, and the group consisting of the oligonucleotides described in SEQ ID NOS: 4, 13, and 14, respectively. Oligonucleotides selected at least one at a time may be used.

[0015] A third invention of the present invention is a composition used for a polymorphism detection method in which detection of VNTR in a target nucleic acid and detection of SNP present in the repetitive unit sequence of the VNTR are simultaneously performed. A labeled oligonucleotide that identifies the SNP present in the repeat unit sequence of the VNTR; an oligo that is hybridized in the boundary region between the most upstream repeat sequence and the upstream sequence adjacent to the repeat sequence The present invention relates to a composition containing an oligonucleotide which is hybridized in the boundary region between a nucleotide and a repetitive sequence which is the most downstream among the repetitive sequences and a downstream sequence adjacent to the repetitive sequence. In the third invention of the present invention, VN

A labeled oligonucleotide that identifies an SNP present in the repeat unit sequence of TR, an oligonucleotide that hybridizes to the boundary region between the most upstream repeat sequence in the repeat sequence and the upstream sequence adjacent to the sequence, And oligonucleotide force hybridizing to the boundary region between the most downstream repeat sequence in the repetitive sequence and the downstream sequence adjacent to the repeat sequence, and the oligonucleotide force set forth in SEQ ID NOS: 1 and 2 in the sequence listing, It may be an oligonucleotide selected from at least one each selected from the group consisting of the oligonucleotides described in SEQ ID NOs: 3 and 12, and the group consisting of oligonucleotides described in SEQ ID NOs: 4, 13, and 14.

[0016] The fourth invention of the present invention is a kit for use in a polymorphism detection method in which detection of VNTR in a target nucleic acid and detection of SNP present in a repetitive unit sequence of the VNTR are simultaneously performed. A labeled oligonucleotide that identifies an SNP present in the repeat unit sequence of VNTR, and is located in the boundary region between the repeat sequence that is most upstream in the repeat sequence and the upstream sequence adjacent to the sequence, The present invention relates to a kit containing an oligonucleotide to be hybridized and an oligonucleotide that hybridizes to the boundary region between the most downstream repetitive sequence and the downstream sequence adjacent to the repetitive sequence. In the third invention of the present invention, a labeled oligonucleotide for identifying an SNP present in a repeat unit sequence of VNTR, a boundary between the most upstream repeat sequence in the repeat sequence and the upstream sequence adjacent to the sequence Oligonucleotides to be hybridized in the region, oligonucleotides to be hybridized in the boundary region between the most downstream repetitive sequence in the repetitive sequence and the downstream sequence adjacent to the repetitive sequence. An oligonucleotide selected from the group consisting of the oligonucleotides described in 2, the group also including the oligonucleotides described in SEQ ID NOs: 3 and 12, and the group consisting of the oligonucleotides described in SEQ ID NOs: 4, 13, and 14; There's ヽ.

The invention's effect

[0017] According to the present invention, it is possible to simultaneously and easily detect VNTR and the SNP typing present in the repeating unit sequence of the VNTR with high sensitivity.

Brief Description of Drawings

FIG. 1 is a diagram showing the results of polymorphism detection by the method for detecting a genetic polymorphism of the present invention.

FIG. 2 is a diagram showing the results of polymorphism detection by the method for detecting gene polymorphisms of the present invention.

FIG. 3 is a schematic diagram showing an example of a method for detecting a gene polymorphism of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0019] In this specification, the variable sequence of tandem repeat (VN TR) is a repetitive sequence in which two or more identical or very similar base sequences are repeated, and the repeat of the base sequence. Indicates a polymorphism with a different number of times. VNTR is also called mini satellite. The base sequence that is the repeating unit is referred to as a repeating unit sequence. Micro-satellite (short tandem repe at: STR) has a relatively short repeat unit sequence. There is no clear distinction between VNTR and STR. Include in VNTR. [0020] In this specification, single nucleotide polymorphism (SNP) means a polymorphism in which the nucleotide sequence of a nucleic acid such as a genome differs by one base between a plurality of individuals.

In the present specification, upstream and downstream indicate a positional relationship with respect to the direction in which RNA is transcribed from a promoter sequence. For example, when the translation start point is used as a reference, the 5 ′ UTR (untranslated region) is upstream and the coding region is downstream.

[0022] In this specification, the simultaneous detection of VNTR and the detection of SNP present in the repetitive unit sequence of the VNTR comprises preparing one reaction solution or composition by mixing a plurality of components, This means that the reaction solution obtained by reacting the preparation is in a state where VNTR and SNP can be detected.

In the present specification, the term “hybridization” means that a single-stranded nucleic acid forms a hybrid (hybrid double-stranded nucleic acid molecule) by complementary base pairing. In the present specification, it means that a nucleic acid is hybridized when an hybrid is formed, and the nucleic acid sequences forming the hybrid may be either completely complementary or partially complementary.

[0024] In this specification, the boundary region means a region (connected portion) that becomes a boundary in two connected specific arrays. Hybridizing to the boundary region means to hybridize all or part of each of the above two specific sequences.

[0025] (1) Method for detecting a polymorphism of a gene of the present invention

The method of the present invention is a method for detecting a polymorphism of a gene characterized by the simultaneous detection of VNTR and SNP present in the repeat unit sequence of the VNTR, comprising the step of simultaneously hybridizing at least three oligonucleotides. Inclusive detection method. The oligonucleotide used in the method of the present invention is not particularly limited as long as VNTR and SNP can be detected simultaneously.

(a) a labeled oligonucleotide that identifies the SNP in the sequence of the repeat unit of the VNTR,

(b) an oligonucleotide that hybridizes to the boundary region between the most upstream repeat sequence in the repeat sequence and the upstream sequence adjacent to the repeat sequence,

(c) a repetitive sequence which is the most downstream among repetitive sequences and a downstream sequence adjacent to the repetitive sequence Oligonucleotides that hybridize to the border region. In the present specification, the description that at least three oligonucleotides are simultaneously hybridized means that the target nucleic acid has been oligonucleotideized in at least three locations. Irrelevant, if at least three sites are connected at the part and nobled, it indicates that there are substantially at least three oligonucleotides.

[0026] (a) The labeled oligonucleotide for identifying the SNP in the sequence of the repeat unit of VNTR is an oligonucleotide cleaved by an enzyme such as endonuclease exonuclease after hybridization, or an oligonucleotide by a DNA polymerase or the like. Any of the labeled oligonucleotides that distinguish SNPs by nucleotide extension reaction can be preferably used. It is also possible to set the SNP site at the 5th, 3rd, 3rd, or end of the oligonucleotide. The method for identifying the SNP is not particularly limited, and for example, the methods of Patent Document 1 to Patent Document 5 can be used.

[0027] When an SNP site is set inside the labeled oligonucleotide, the SNP can be identified by hybridization with a target VNTR repetitive sequence and then cleavage with a mismatch-sensitive nuclease. For example, when the target VNTR repeat sequence and the labeled oligonucleotide are completely hybridized, a restriction enzyme that cleaves the SNP site, nicking endonuclease (manufactured by NEB), and a cleavase that recognizes a specific DNA structure (see Patent Document 5) Can be used. The SNP is identified by detecting the labeled oligonucleotide cleaved by the nuclease. If you want to avoid cleavage of the target nucleic acid to which the labeled oligonucleotide hybridizes, you can prevent cleavage by the nuclease by methylating the target nucleic acid, using a nucleic acid analog, etc. (Nelson M, 2 others, Nucleic Acids Res 1993, vol. 21, p. 3139-54, Sayers J, et al., Nucleic Acids Res. 1989, vol. 17, p. 9495). Furthermore, ribonuclease H can be preferably used since the SNP site of the oligonucleotide is a ribonucleotide. In this case, the SNP site and the sequence before and after it as ribonucleotides, and other nucleotides as deoxyribonucleotides, match or mismatch the SNP sites of chimeric oligonucleotides or the sites before and after them. Can be cut according to the SNP The Ribonuclease H may be a commercially available enzyme. Heat-resistant ribonuclease H can be prepared by the method described in WO 02Z22831. A mismatch-specific nuclease can also be used when mismatches occur between the target VNTR repeat and the labeled oligonucleotide (Smith J, et al., Proc. Natl. Acid. Sci. USA). , 1996, vol. 93, p. 4374—9).

The hybridization of the labeled oligonucleotide to the target nucleic acid is determined by the length of the oligonucleotide, the reaction solution, and the temperature. The reaction solution and temperature may be appropriately set as long as the enzyme used can maintain the activity. Depending on the reaction solution and temperature set, a labeled oligonucleotide having an appropriate length can be designed to allow and hybridize with the target nucleic acid. By calculating the Tm of the designed oligonucleotide and comparing it with the reaction temperature, it can be predicted whether or not the target nucleic acid will be hybridized. In general, the Tm of an oligonucleotide can be obtained by, for example, the following formula.

Tm = 81. 5- 16. 6 (log [Na ⁺ ]) +0.41 (% G + C)-(600 / N)

Ten

(Where N is the length of the oligonucleotide, and% G + C is the content of guanine and cytosine residues in the oligonucleotide probe or primer.) The length of the oligonucleotide is 18 bases. When shorter, Tm is, for example, the sum of the product of A + T (adenine + thymine) residue and 2 ° C and the product of G + C residue and 4 ° C [ (A + T) X 2+ (G + C) X 4]

When the SNP site is set inside, the labeled oligonucleotide may have a chain length that hybridizes to the target nucleic acid, cleaves with the enzyme, and then releases the target nucleic acid force. In this case, another labeled oligonucleotide molecule repeats hybridization with the target nucleic acid and the detection sensitivity is improved, which is suitable for the method of the present invention. The smaller the Tm of the oligonucleotide fragment after cleavage, compared to the reaction temperature, the more the target nucleic acid force is released. The chain length of the labeled oligonucleotide after cleavage is not particularly limited. For example, it is 3 to 40 bases, preferably 4 to 25 bases, more preferably 5 to 20 bases.

When an SNP site is set at the 5 'end of the labeled oligonucleotide, after hybridization to the target VNTR repeat sequence, the free nucleic acid generated by, for example, cleavage of the 5' end with a DNA polymerase having 5 'exonuclease activity is used. By detecting the label SNPs can be identified. For example, the detection method of the present invention can be carried out by using the TaqMan probe described in Patent Document 2 as a labeled oligonucleotide for detecting SNP and detecting the degradation of the 5 ′ end of the probe.

[0029] When an SNP site is set at the 3 'end of the labeled oligonucleotide, after hybridizing to the target VNTR repeat sequence, the presence or absence of incorporation of the 3' end base by polymerase reaction using the target sequence as a saddle type SNPs can be identified. It is also possible to set the 3 'end of the labeled oligonucleotide to the base next to the SNP site of the target sequence and identify the SNP by the base of the SNP site incorporated by polymerase reaction or the like. For example, the primer described in Patent Documents 3 and 4 is used as a labeled oligonucleotide for detecting SNP, and the detection method of the present invention is carried out by detecting the extension reaction of the 3 ′ end of the primer. can do.

[0030] A plurality of labeled oligonucleotides for identifying SNPs may be used depending on the SNP. The label can be selected according to the base of each SNP. Furthermore, a label can be appropriately selected according to the design of the labeled oligonucleotide, the nuclease used, and the detection method. The labeling substance that can be used in the present invention is not particularly limited as long as the SNP can be identified. For example, labeling by interaction of piotin, avidin, antigen, antibody, etc., radioisotope, dye, fluorescent substance, etc. It may be a sign by detection of a signal. In particular, in the case of a method for discriminating SNPs by nuclease cleavage, it is preferable to label each molecule generated by cleavage with a combination of different fluorescent substances or fluorescent substances and quenching substances. 6-FAM (6-carboxyfluoresce in) and DABCYL (4-mmethylaminoazobenzene—4'-sulfone), ROX (6-carboxy-X-rho damine) and DABCYL, 6-FAM and Eclipse (Epoch) Biosciences), ROX and Eel ipse, TET (tetrachlorofluorescein) and DABCYL, TET and Eclipse, etc. can be suitably used. Furthermore, HEX (hexachlorofluorescein) can also be preferably used.

[0031] (b) The oligonucleotide that is hybridized in the boundary region between the most upstream repetitive sequence in the repetitive sequence and the upstream sequence adjacent to the repetitive sequence is the most upstream repetitive sequence and the sequence. A labeled oligonucleotide that hybridizes to both adjacent upstream sequences and identifies the SNP in the sequence of the repeat unit of the VNTR to the most upstream repeat sequence. And oligonucleotides that inhibit or compete with hybridization.

(C) The oligonucleotide that is hybridized in the boundary region between the most downstream repetitive sequence in the repetitive sequence and the downstream sequence adjacent to the repetitive sequence is the most downstream repetitive sequence and the sequence. A labeled oligonucleotide that hybridizes to both adjacent downstream sequences and identifies the SNP in the sequence of the repeat unit of the VNTR to the most downstream repetitive sequence, inhibits or competes with hybridization.

[0033] The oligonucleotides used in the present invention, that is, the oligonucleotides (a) to (c) described above are modified so that extension from the 3 ′ end by a polymerase or the like does not occur as necessary. Also good. Modification may be performed when an enzyme that catalyzes an extension reaction, such as DNA polymerase, is present in the reaction solution and an extension reaction from an oligonucleotide is not desired. For example, it is possible to add a dideoxynucleotide at the 3 ′ end, a nucleotide modified with the hydroxyl group at the 3-position of ribose, or a nucleotide that has been modified such that extension by DNA polymerase is hindered by steric hindrance. can give. Alkylation, amidation, amination and other known modification methods can be used as a method for modifying the hydroxyl group at the 3-position of the ribose of the above nucleotide. For example, DNA elongation reaction can be achieved by aminoalkylation. Can be prevented. As long as the above-mentioned properties can be obtained, the modification may be located at the end of 3, or at the end of the oligonucleotide!

[0034] Furthermore, the oligonucleotide used in the present invention may be modified so that the 5 'end thereof is not cleaved by exonuclease, if necessary! If there is an enzyme that catalyzes the cleavage or degradation reaction, such as DNA polymerase with 5 '→ 3, exonuclease activity, in the reaction solution, the oligonucleotide 5' end should be modified if desired. . The modification is not particularly limited. For example, dephosphorylation at the 5 ′ end, modification of the hydroxyl group at the 3-position of ribose, (α-S) nucleotide in which the oxygen atom bonded to the phosphate group is substituted with a sulfur atom Use, peptide nucleic acid (PNA, peptide nucleic acid; Nature, 365, 566- 568 (1993)), aminoalkylation, dye, fluorescent substance, luminescent substance, quencher, various ligands (piotin, digoxigenin, etc.) · Addition of enzymes, etc., introduction of substituents that may cause steric hindrance. As long as the modification can acquire the above-mentioned properties, the oligonucleotide may be located at the 5, terminal! /, Or 5, terminal /! Oligones The synthesis of the nucleotides is usually entrusted by the company that synthesizes the oligonucleotides, and the company that commissions the synthesis of the oligonucleotides provides the modification of the above-mentioned oligonucleotides at the 3, terminal, 5, and terminal ends as a normal option menu. The

[0035] The oligonucleotide used in the present invention is preferably complementary to the sequence of the target nucleic acid in hybridization with the target nucleic acid, and a part of the sequence may be complementary. Furthermore, the complementary sequence of the oligonucleotide used in the present invention may be localized within the oligonucleotide or scattered as long as it is hybridized. Hybridization conditions such as the length of the oligonucleotide, the composition of the base, the composition of the solution containing the oligonucleotide, and the temperature can be determined based on known knowledge in the art. Although not particularly limited, for example, chapters 8 to 11 of 2001, published by Cold Spring Harbor Laboratories, Sambrook J et al., Molecular Cloning: Laboratory Manual 3rd ed. The hybridization conditions described in 14 and 14 and the PCR reaction conditions may be referred to when the nucleic acid amplification reaction is performed simultaneously. In particular, buffers recommended by commercially available real-time PCR systems, reaction conditions, and oligonucleotides that hybridize under such conditions can be used preferably. Furthermore, the oligonucleotide used in the present invention may be one oligonucleotide that hybridizes to a plurality of repeating units across the boundary region between the repeating unit of the V NTR and the repeating unit in the target nucleic acid. A single oligonucleotide that does not hybridize to multiple repeat units across the boundary region between repeat units and repeat units.

[0036] The length of the oligonucleotide used in the present invention is an appropriate length depending on the length of the VNTR repeat unit of the target gene, the enzyme used, the type of label when labeling, and the hybridization conditions. The length is not particularly limited as long as it is selected. For example, the length is 6 to 80 bases, preferably 8 to 50 bases, more preferably 10 to 40 bases.

[0037] In the method for detecting a genetic polymorphism of the present invention, the above-mentioned oligonucleotides (b) and (c) are hybridized to the most upstream repeat sequence and the most downstream repeat sequence among the repeat sequences of VNTR. Soybeans. Therefore, if the labeled oligonucleotide (a) is hybridized, it is a VNTR that repeats S3 or more times if it is hybridized, and if it is not hybridized, the repeated sequence is repeated twice. Or VNTR that does not repeat. In other words, if there is no repetitive sequence, or if it is a single repeat, the labeled oligonucleotide (a) will not hybridize, and if it is repeated three times, the labeled oligonucleotide (a) will be located at one location in the target sequence. No, I'll give it. Similarly, in the case of 4 repetitions, the labeled oligonucleotide of (a) hybridizes to 2 positions of the target sequence, and in the case of 5 repetitions, it hybridizes to 3 positions. Furthermore, the SNP of the repetitive sequence other than the most upstream repetitive sequence and the repetitive sequence other than the most downstream repetitive sequence among the VNTR repetitive sequences to which the labeled oligonucleotide (a) hybridizes can be distinguished at the same time. For example, in the case of 3 repeats, the SNP of the middle repeat can be identified. In addition, when the labeled oligonucleotide ( _a ) is hybridized at multiple locations in the target sequence, it is possible to classify the type of SNP by preparing multiple labeled oligonucleotides (a) according to the SNP. Noh. That is, the gene polymorphism detection method of the present invention includes the typing of a specific polymorphism, the detection of being any one of a plurality of types, and the confirmation of V being not a specific type. Is done.

[0038] In the method of the present invention, the detection of the above-mentioned VNTR and SNP can be performed in one reaction system and one reaction vessel. During the detection reaction, reagents are added, and reaction products are purified, separated, and extracted. This is a method with few accidents such as contamination, which requires work such as changing the reaction system and exchanging the knocker. Furthermore, since the labeled oligonucleotide (a) is competitively and hybridized with the oligonucleotides (b) and (c) above, it is not necessary to set and control strict hybridization conditions. Is characterized by high.

[0039] The gene polymorphism detection method of the present invention can rapidly detect the polymorphism. The time required for detection of VNTR and SNP of the present invention is not particularly limited, but it is within 12 hours, preferably within 6 hours, more preferably within 3 hours, even more preferably within 2 hours, particularly preferably. Within 90 minutes.

[0040] The gene polymorphism detection method of the present invention can detect the polymorphism with high sensitivity. If there are alleles in the target gene and the polymorphism type is heterogeneous, the sample containing the target nucleic acid will contain multiple types. The method for detecting a gene polymorphism of the present invention is not particularly limited, but the type of a sample present at 50%, preferably 30% or more, more preferably 10% or more, and further preferably 5% or more is not limited. This is a highly sensitive detection method.

[0041] In the method for detecting a gene polymorphism of the present invention, any gene can be used as a target gene as long as it is a polymorphism suspected of having SNP in the VNTR repeat unit sequence. Although not particularly limited, for example, when detecting a polymorphism in the 5 ′ UTR region of a thymidylate synthase (TS) gene, a VNTR repeat unit (SEQ ID NO: 7) consisting of 28 bases is used. Labeled oligonucleotide that identifies SNPs (C and G of the 12th base) present in the sequence, in the boundary region between the most upstream repeat sequence in the repeat sequence and the upstream sequence adjacent to the sequence Use an oligonucleotide that hybridizes, an oligonucleotide that hybridizes to the border region between the most downstream of the repetitive sequence and the downstream sequence adjacent to the repetitive sequence.

[0042] Hereinafter, in this specification, the genotype of the 5 'UTR polymorphism of the TS gene is defined as follows. 2R has 2 repeat units (SEQ ID NO: 7) in the VNTR, 2R G is SNP in order of upstream force (that is, the 12th base in SEQ ID NO: 7 is G), G gene Indicates the type. 2RC indicates a genotype in which SNP also has upstream force G and C (that is, the 12th base in SEQ ID NO: 7 is C). 3R has 3 repeats in VNTR, and 3RG shows G, G, and C genotypes from upstream of SNP. 3RC shows G, C, and C genotypes in which SNP also has upstream force.

[0043] Detection of the TS gene 5 'UTR polymorphism using the method of the present invention is not particularly limited. For example, a labeled nucleotide that identifies C of SNP and another labeled oligonucleotide that identifies G of SNP are used. Then, VNTR of 2R and 3R or higher can be distinguished by hybridization of these labeled oligonucleotides. That is, if either of the above labeled nucleotides is hybridized, it indicates that the VNTR is 3R or higher, and if not hybridized, it indicates that there is no 2R VNTR or VNTR. Furthermore, C and G SNPs can be discriminated by the signal from the labeled oligonucleotide. That is, in the case of 3R, 3RC can be discriminated by a labeled nucleotide that identifies 3RG force C by a labeled nucleotide that identifies G. In the case of 4R, if only the signal of the labeled nucleotide that identifies G is the signal of the middle two, the repeat sequence in the middle is GG, and if only the signal of the labeled nucleotide that identifies C is the signal, the middle two repeat sequences are CC, Two repeats in the middle if both labeled nucleotide signals are detected Indicates that the sequence is GC or CG. For example, if there is a possibility that 2RG, 2RC, 3RG, 3RC type nucleic acid is present in the sample, for example, if a labeled oligonucleotide signal that identifies G of SNP is obtained, 3RG type nucleic acid is obtained. Can be detected. Since the 3RG polymorphism of the TS gene and polymorphisms of 4R or higher greatly affect the sensitivity and efficacy of the anticancer agent 5-FU, 5—1; 31 ^} polymorphism is detected before treatment, and the gene This is very useful.

[0044] When detecting a polymorphism in the 5 'UTR region of the TS gene, although not particularly limited, for example, among the labeled oligonucleotides that identify the SNP in the sequence of the repeat unit of VNTR, identify the SNP of G As a labeled oligonucleotide, an oligonucleotide having the sequence of SEQ ID NO: 1 labeled with FAM at the end, 3, an end force of ¾clipse as a labeled oligonucleotide, a SNP identifying a C SNP, and a 5 ′ end as ΗΕΧ, 3 ′ As an oligonucleotide having the sequence described in SEQ ID NO: 2 labeled with terminal force ¾clipse, an oligonucleotide that hybridizes to the border region between the most upstream repeat sequence in the repeat sequence and the upstream sequence adjacent to the sequence, An oligonucleotide having the sequence of SEQ ID NO: 3 or 12 modified at the 3 ′ end by amination, a repetitive sequence at the most downstream of the repetitive sequences, and the As oligonucleotides Haiburidizu the boundary region between its downstream sequence flanked beside the column, 3 'end oligonucleotide can be preferably used having the sequence of the modified SEQ ID NO: 4, 13 or 14, wherein the amination. In this case, the labeled oligonucleotide that identifies the SNP in the sequence of the VNTR repeat unit hybridizes to the target nucleic acid, and the change in wavelength associated with the energy transfer of the fluorescent substance is detected by cleavage of the oligonucleotide with ribonuclease H. Thus, the polymorphism can be detected. The method for detecting a polymorphism in the 5 ′ UTR region of the TS gene of the present invention is not particularly limited, but a schematic diagram is shown in FIG. 3 as an example. In Figure 3, when the target nucleic acid polymorphism is 3RG, the label of the labeled oligonucleotide (labeled G) that identifies the SNP of G is detected (+). In the case of 3RC, the label that identifies the SNP of C The oligonucleotide label (label C) is detected (+).

[0045] In the method for detecting a gene polymorphism of the present invention, simultaneously with the detection of the polymorphism, nucleic acid amplification of a region where the polymorphism exists may be performed. By simultaneously amplifying the nucleic acid, the detection sensitivity of the polymorphism can be improved. As a nucleic acid amplification method, a known method can be used. For example, polymerase chain reaction (PCR; polymerase chain reaction, US Pat. No. 4,683,195), strand displacement amplification (SDA; strand displacement amplification, JP7-17-1718), ICAN (Isothermal and Nucleic acid amplification methods such as Cnimeric primer-initiated Amplification of Nucleic acids (WO 00/56877 pamphlet) can be used.

Nucleic acid amplification reaction can be carried out according to the attached procedure by using a knoffer attached to a commercially available product.

[0046] When detecting a polymorphism in the 5 'UTR region of the TS gene, although not particularly limited, for example

PCR reaction using a primer having the sequence of SEQ ID NO: 5 and a primer having the sequence of SEQ ID NO: 6 is preferred.

[0047] The gene polymorphism detection method of the present invention can be carried out at any specific time point during the reaction or after the reaction end, in which real-time detection for detecting the signal of the label continuously or intermittently can be performed. Endpoint detection may be performed.

[0048] (2) Composition and kit for detecting the gene polymorphism of the present invention

The compositions and kits of the present invention are present in VNTR detection and repeat units of the VNTR

It is a composition and kit containing at least the following three oligonucleotides, which can be used in a method for detecting a polymorphism of a gene characterized by performing SNP detection simultaneously.

(c) an oligonucleotide that hybridizes to the boundary region between the most downstream repetitive sequence and the downstream sequence adjacent to the repetitive sequence.

[0049] The composition and kit of the present invention can be used in the method of the present invention described in (1). It may also contain primers for amplifying the nucleic acid in the VNTR region of the target gene. Furthermore, it may contain a label as a standard substance such as an enzyme such as DNA polymerase, restriction enzyme, cleavase, endonuclease, exonuclease, ribonuclease H, or fluorescent dye. The kit of the present invention may contain instructions. The “instruction” refers to the method of using the kit, for example, the method of preparing a reagent solution, recommended reaction conditions, etc. This printed matter includes printed manuals in the form of brochures or leaflets, as well as labels attached to kits and packages containing kits. Furthermore, the information disclosed and provided through an electronic medium such as the Internet is included. The composition of the present invention may contain a reaction buffer. The reaction buffer used in the present invention may be appropriately selected according to a method for detecting SNP, a method for detecting a label signal, and a method for amplifying nucleic acid. These methods often use enzymes derived from microorganisms such as Escherichia coli, and there are many common points in the environment where the reaction proceeds in general, so a common reaction solution in which each reaction proceeds is used. It is easy to set. Although not particularly limited, for example, the reaction buffer used in the present invention is one containing a buffer component, a magnesium salt or other metal salt, or dNTP. In addition, it is natural to optimize the salt type and concentration according to the metal requirements of the enzyme used. The buffer component is not particularly limited, and for example, bicine, tricine, hepes, tris, phosphate (sodium phosphate, potassium phosphate, etc.) can be preferably used. In particular, a buffer containing bicine, tricine, hepes, or phosphate as a buffer component is suitable for the present invention. Although there is no particular limitation, for example, when the reaction temperature is high, the pH change due to temperature change is small, the bicine buffer solution is preferred, and depending on the type of RNaseH used, the Hepes buffer solution may be It may be preferable. Therefore, an optimal buffer may be selected depending on the reaction temperature, DNA polymerase or endonuclease used. The final concentration of the buffer component is in the range of 5 mM to: LOO mM, particularly preferably in the range of 10 mM to 50 mM, and in the range of pH 6.0 to 9.5, particularly preferably ί or pH 7.0 to 9.2. Power S used. In the case of containing a magnesium salt, although there is no particular limitation, for example, magnesium chloride, magnesium acetate, or magnesium sulfate can be suitably used. It is. In addition, when dNTPs (dATP, dCTP, dGTP, dTTP mixture), which are substrates for DNA elongation reaction, are included, the final concentration is in the range of 0. 1 lmM to 3. OmM, particularly preferably 0.2 mM to l.2 mM, respectively. It is. The amount of the oligonucleotide used is in the range of 0.01 μ 0 to 10 μ 、, and particularly preferably in the range of 0.05 μ 0 to 1 μΜ. In addition, amplification reaction in the reaction solution. Additives for the purpose of stabilization, etc. can coexist, with final concentrations of 0.1% or less urine serum albumin (BSA), 10% or less dimethyl sulfoxide (DMS 0), 4 mM or less Putrescine dihydrochloride or propylene diamine of 0.01% or less may be added. In addition, NMP (1-methyl 2-pyrrolidinone), glycerol, polyethylene glycol, dimethyl sulfoxide, and Z or formamide may be included. By adding these organic solvents, non-specific annealing of the oligonucleotide is lightened. Expected to be reduced.

When using an endonuclease, for example, RNaseH derived from E. coli is preferably in the range of 3 to 200 U per 50 μl of reaction solution, particularly in the range of 151; to 60 U. Similarly, in the case of RNa seH derived from Pyrococcus bacteria or Alkaeoglobus bacteria, it is in the range of 3 to 200 U, more preferably in the range of 5 to 50 U per 50 / zL of reaction solution. Further, if the DNA polymerase is, for example, BcaBEST DNA polymerase (manufactured by TAKARA BIO INC.), The range of 0.51 per reaction solution volume 50 1; to the range of 100 U, particularly 1 U to 22 U is preferable.

In the present invention, when endonuclease and DNA polymerase are combined, there is no particular limitation. For example, a combination of RNaseH and BcaBEST DNA polymerase derived from Escherichia coli, Pyrococcus bacteria, or Arcaerobus bacteria is preferable. Furthermore, it is expected that the number of units that can be suitably used varies depending on the type of endonuclease and DNA polymerase. In that case, it is only necessary to adjust the composition of the knofer used and the amount of the enzyme added, using the detection sensitivity improvement or the amount of the amplified product as an index. In either case, it is natural to optimize the composition of the reaction buffer according to the type of enzyme used.

The composition and kit of the present invention can be used in a method for detecting a polymorphism of any gene having a polymorphism in which SNP is suspected to exist in the sequence of the repeat unit of VNTR. Although not particularly limited, for example, when detecting a polymorphism in the 5 ′ UTR region of the thymidylate synthase (TS) gene, the composition and kit of the present invention have a V NTR of 28 bases. A labeled oligonucleotide that identifies SNPs (C and G of the 12th base) present in the sequence of the repeat unit (SEQ ID NO: 7), and the repeat sequence most upstream of the repeat sequence Oligonucleotides that hybridize in the boundary region with the upstream sequence adjacent to the column, oligonucleotides that hybridize to the boundary region between the most downstream repetitive sequence in the repetitive sequence and the downstream sequence adjacent to the sequence Containing.

[0051] In the case of the composition and kit for detecting a polymorphism in the 5 'UTR region of the TS gene, for example, without limitation, for example, a labeled oligonucleotide that identifies the SNP in the sequence of the repeating unit of VNTR. Among them, as a labeled oligonucleotide for identifying the SNP of G, an oligonucleotide having the sequence described in SEQ ID NO: 1, labeled with FAM at the end, and Eclipse at the 3 ′ end, as a labeled oligonucleotide for identifying the SNP of C, 5. An oligonucleotide having the sequence of SEQ ID NO: 2 labeled with HEX at the end, labeled with Eclipse at the end, and an upstream sequence adjacent to the sequence and the upstream sequence adjacent to the sequence. As an oligonucleotide that hybridizes to the boundary region, the oligonucleotide having the sequence described in SEQ ID NO: 3 or 12 modified at the end by amination and the most of the repetitive sequences As an oligonucleotide that hybridizes to the boundary region between a downstream repetitive sequence and a downstream sequence adjacent to the sequence, the sequence described in SEQ ID NO: 4, 13, or 14 whose 3 ′ end is modified by amination is used. The oligonucleotide having is preferred.

[0052] Further, the composition and kit of the present invention may contain a primer for amplifying the nucleic acid of the 5 'UTR region of the TS gene. A primer having a sequence and a primer having the sequence described in SEQ ID NO: 6 may be contained.

Example

[0053] The ability to describe the present invention more specifically with reference to the following examples. The present invention is not limited to the following examples.

Among the operations described in this specification, the basic operations are published in 2001 by Cold Spring Harbor Laboratory, edited by T. Maniatis et al., Molecular Cloning: Laboratory Laboratory. According to the method described in the third edition (Molecular Cloning: A Laboratory Manual 3rd ed.).

[0054] Example 1 TS 5, one UTR VNTRZSNP detection

5. Orientation labeled with FAM at the end and 3, with eclipse (epoch bioscience) at the end. Oligonucleotide VN2G (SEQ ID NO: 1), 5, end labeled with HEX, 3, end labeled with eclipse VN2C (SEQ ID NO: 2), oligonucleotide VN1 (SEQ ID NO: 3), oligonucleotide VN3 (SEQ ID NO: 4 ), Primer 1F (SEQ ID NO: 5), primer 2R (SEQ ID NO: 6) were prepared using a DNA synthesizer.

To evaluate the detection system, use standard genomic DNA with genotypes of 3RG3RG (ie alleles are all 3RG, and so on), 2RG3RG, 2RC3RG, 3RG3RC, 2RC3RC, 2RG3RC, 3RC3RC, 2RG2RC, 2RC2RC It was. The standard genomic DNA was prepared from human blood with informed consent according to the method of Cancer Res. 2003, vol. 63, p. 6004-7 (Non-patent Document 5), and Haelll was used. The VNTRZSNP dienotype of TS 5 ′ UTR was determined by PCR-R FLP method. The sequence of the VNTRZSNP region of 3RG in SEQ ID NO: 8, 3RC in SEQ ID NO: 9, 2RG in SEQ ID NO: 10, 2 RC in SEQ ID NO: 11 is shown.

Ribonuclease H derived from Thermococcus litoralis (Tli) was prepared by the method described in WO 02/22831.

GC buffer Π (Takara Bio), 0.4 mM dNTPs, 0.2 ^ M primer IF, 0.2 μM primer 2R, 0.1 ^ M VN2G, 0.1 ^ M VN2C, 1.6 M VN1, 1 A 25 μ1 amplification reaction consisting of 6 μΜ VN3, 1% ROX Reference Dye (Invitrogen), 1. 25 U TaKaRa LA Taq (Takara Bio), 50 U Tli RNaseH II (Takara Bio), lOOng standard genomic DNA Created. PCR amplification and detection were performed using a 7500 system (Applied Biosystems). PCR reaction conditions are 95. C, 15 seconds, 60. C, 40 seconds, 72. C, 45 cycles of 30 seconds were performed. The fluorescence of FAM and HEX was measured by ABI7500 system. The results are shown in Fig. 1. Figure 1 shows a graph plotting the FAM fluorescence signal from VN2G on the vertical axis and the HEX fluorescence signal from VN2C on the horizontal axis for each genotype. As a result, an increase in the fluorescence signal of FAM by VN2G was detected in genotypes with 3RG of 3RG3RG (3G3G in the figure), 2RG3RG (2G3G in the figure), 2RC3RG (2C3G in the figure), and the fluorescence signal of HEX by VN2C was detected. There was no increase. For genotypes with 3RC: 2RC3RC (2C3C in the figure), 2RG3RC (2G3C in the figure), 3RC3RC (3C3C in the figure), the HAM firefly by VN2C An increase in the light signal was detected, and no increase in the fluorescence signal of FAM by VN2G was observed. In addition, in the 3RG3RC (3G3C in the figure) genotype, both oligonucleotides increased FAM and HEX fluorescence. In the 2RG2RC (2G2C in the figure) and 2RC2RC (2C2C in the figure) genotypes that do not have the 3R genotype, there was a strong increase in both fluorescence signals. As shown in Fig. 1, the 3RG and 3RC genotypes of TS 5 'UTR VNTRZSNP could be clearly determined by this method.

[0056] Example 2 TS 5'—Detection sensitivity of UTR VNTRZSNP

In order to determine the detection sensitivity of 3RG using this method, genomic DNA with the 3RG3RG genotype is diluted with genomic DNA with the 3RC3RC genotype to create 100, 50, 25, 10, 5% 3RG3RG genomic solutions. did. In the same manner as in Example 1, 3RG was detected using a Rotor Gene 2000 (Corbett Research). The result is shown in Fig.2. As shown in Figure 2, an increase in the fluorescence signal of FAM by VN2G was also detected in the 5% 3RG3RG genomic solution, and the fluorescence signal was significantly higher than that of the 100% 3RC3RC (0% in Figure 2) genomic solution. Obtained. Therefore, it was shown that this method is a highly sensitive detection system that can detect even contamination of 5% 3RG genome.

[0057] Example 3 Examination of oligonucleotides

In Example 1, VN1.2 (SEQ ID NO: 12) was used instead of VN1, VN3.2 (SEQ ID NO: 13), and VN3.3 (SEQ ID NO: 14) were used instead of VN3. went . As a result, the genotype could be determined in the same manner as in Example 1.

Industrial applicability

[0058] According to the present invention, a method for simultaneously and simply detecting VNTR and SNP typing with high sensitivity and a kit for use in the method are provided.

Sequence listing free text

[0059] SEQ ID N〇: l: Chimeric oligonucleotide designated as VN2G. Nucleotide 4 is nbo nucleotide- other nucleotides are deoxyribonucleotides "

SEQ ID NO: 2: Chimeric oligonucleotide designated as VN2C. "Nucleotide 4 is ribo nucleotide— other nucleotides are deoxyribonucleotides" SEQ ID NO: 3: Oligonucleotide designated as VNl.SEQ ID NO: 4: Oligonucleotide designated as VN3.SEQ ID NO: 5: Oligonucleotide primer designated as IF, SEQ ID NO: 6: Oligonucleotide primer designated as 2R, SEQ ID NO: 12: Oligonucleotide designated as VNl. 2.SEQ ID NO: 13: Oligonucleotide designated as VN3.2. SEQ ID NO: 14: Oligonucleotide designated as VN3.3.

Claims

Claims [1] Polymorphism characterized in that detection of repetitive sequence polymorphism (VNTR) in the target nucleic acid and single nucleotide polymorphism (SNP) present in the repeat unit sequence of the VNTR are simultaneously performed. A detection method comprising the step of simultaneously hybridizing a target nucleic acid for detecting a polymorphism and at least three oligonucleotides. [2] Three oligonucleotides

(3) an oligonucleotide that hybridizes to the boundary region between the most downstream repetitive sequence in the repetitive sequence and the downstream sequence adjacent to the repetitive sequence;

The method for detecting a polymorphism according to claim 1, wherein

[3] The method for detecting a polymorphism according to claim 1 or 2, wherein the hybridization is performed simultaneously with the amplification reaction of the target nucleic acid for detecting the polymorphism.

[4] The following steps:

(2) A step of subjecting the reaction solution prepared in step (1) to a reaction for amplifying a target nucleic acid for polymorphism detection; and

A method for detecting a polymorphism, comprising simultaneously detecting VNTR in a target nucleic acid including, and detecting SNP present in the repeating unit sequence of the VNTR.

[5] The polymorphism detection according to any one of claims 1 to 4, wherein the target nucleic acid for polymorphism detection is a nucleic acid corresponding to the 5 'untranslated region of the thymidylate synthase (TS) gene. Method.

[6] From the group consisting of the oligonucleotides described in SEQ ID NOS: 1 and 2 in the Sequence Listing, the group consisting of the oligonucleotides described in SEQ ID NOS: 3 and 12, and the group consisting of the oligonucleotides described in SEQ ID NOS: 4, 13, and 14, respectively. 6. The method for detecting a polymorphism according to claim 5, wherein oligonucleotides selected at least one by one are used.

[7] A composition used in a polymorphism detection method for simultaneously detecting VNTR in a target nucleic acid and detecting SNP present in the repeat unit sequence of the VNTR, wherein the composition is used in the repeat unit sequence of VNTR. A labeled oligonucleotide that identifies the SNP present in the oligonucleotide, the oligonucleotide that hybridizes to the border region between the most upstream repeat sequence and the upstream sequence adjacent to the repeat sequence, the most downstream of the repeat sequences A composition comprising an oligonucleotide that is hybridized in a boundary region between a repetitive sequence in the sequence and a downstream sequence adjacent to the sequence.

[8] Labeled oligonucleotide that identifies SNPs present in the repeat unit sequence of VNTR, hybridizes to the boundary region between the most upstream repeat sequence and the upstream sequence adjacent to the repeat sequence Oligonucleotide and oligonucleotide force S to be hybridized in the boundary region between the most downstream repetitive sequence in the repetitive sequence and the downstream sequence adjacent to the repetitive sequence, as described in SEQ ID NOs: i and 2 in the sequence listing 8. An oligonucleotide selected from at least one each selected from the group consisting of oligonucleotides, the group consisting of oligonucleotides described in SEQ ID NOs: 3 and 12, and the group consisting of oligonucleotides described in SEQ ID NOs: 4, 13, and 14. The composition as described.

[9] A kit used in a polymorphism detection method that simultaneously detects VNTR in a target nucleic acid and detects SNPs present in the repeat unit sequence of the VNTR, and is present in the repeat unit sequence of VNTR. A labeled oligonucleotide that identifies the SNP to be used, an oligonucleotide that hybridizes to the boundary region between the most upstream repeat sequence in the repeat sequence and the upstream sequence adjacent to the repeat sequence, and most downstream in the repeat sequence A kit comprising an oligonucleotide that hybridizes to a boundary region between a repetitive sequence and a downstream sequence adjacent to the repetitive sequence. A labeled oligonucleotide that identifies an SNP present in the repeat unit sequence of the VNTR, an oligonucleotide that hybridizes to the boundary region between the repeat sequence located most upstream in the repeat sequence and the upstream sequence adjacent to the repeat sequence, A group consisting of oligonucleotides described in SEQ ID NOS: 1 and 2 in the sequence listing, respectively, and the oligonucleotide power to hybridize to the boundary region between the most downstream repeated sequence in the repetitive sequence and the downstream sequence adjacent to the sequence. The kit according to claim 9, wherein the kit is an oligonucleotide selected from at least one each of the group consisting of the oligonucleotides described in SEQ ID NOs: 3 and 12 and the group consisting of the oligonucleotides described in SEQ ID NOs: 4, 13, and 14.