Method for determining CYP2A6 gene polymorphism

Abstract

To develop a method for determining a genotype more accurately than the conventional methods about CYP2A6*4 and / or CYP2A6*7 and establish a measurement system.SOLUTION: In the inventive determination method, two stages of DNA amplification (preliminary amplification and TaqMan-based amplification) are performed, and different characteristic primer sets and probes are used in the case of determining CYP2A6*4 and in the case of determining CYP2A6*7.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for determining CYP2A6 gene polymorphism. [Background technology]

[0002] CYP2A6 is an enzyme involved in the metabolism of nicotine in the body, and is also involved in the metabolism of letrozole (an aromatase inhibitor / postmenopausal breast cancer treatment). There are many alleles of CYP2A6, but in Japanese people, the gene frequencies of the mutant forms CYP2A6*4, which is a gene deletion type, CYP2A6*7 and CYP2A6*10, which have single nucleotide polymorphisms (SNPs) associated with amino acid mutations, and CYP2A6*9, which has an SNP in the promoter region, are high (2-20%). When two of these genotypes are combined, individuals become poor metabolizers (PMs) of nicotine metabolism (Non-Patent Document 1). It is important to identify the genotype that causes PM in Japanese people, but the human genome contains another gene (CYP2A7) near the CYP2A6 locus that has a base sequence very similar to CYP2A6, making it difficult to determine the genotype. A method for determining CYP2A6 gene polymorphisms by melting curve analysis has been reported (Patent Document 1). This method allows amplification of the target gene site and detection of the mutation site to be carried out simultaneously within a single capillary. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Laid-Open No. 2004-194598 [Non-patent literature]

[0004] [Non-Patent Document 1] Nakajima, M. (2006). Nicotine metabolism and CYP2A6 gene polymorphism. JPN.J.ELECTROCARDIOLOGY Vol.26 No.3, 26(3), 217-222. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to develop a method for determining the genotype of CYP2A6*4 and / or CYP2A6*7 with higher accuracy than conventional methods, and to establish a measurement system. [Means for solving the problem]

[0006] The inventors discovered that CYP2A6*4 and / or CYP2A6*7 can be determined with high accuracy by using newly designed PCR primers and hydrolysis probes to perform preprocessing that amplifies only the regions that characterize CYP2A6*1 (wild type), CYP2A6*4, and CYP2A6*7, without amplifying CYP2A7, a separate gene, and thus arrived at the present invention.

[0007] That is, the present invention relates to the following inventions: [1] A method for determining at least one of CYP2A6*4 and CYP2A6*7, which are alleles of CYP2A6, comprising: When determining CYP2A6*4, (1) a step of performing preliminary amplification using a primer set that simultaneously amplifies the regions that characterize CYP2A6*1 and CYP2A6*4 but does not amplify the CYP2A7 gene; (2)(a) a template DNA obtained by diluting the amplification product obtained in the step (1); (b) a primer set capable of simultaneously amplifying a region of 200 bp or less containing a sequence characteristic of CYP2A6*1 and CYP2A6*4; (c) a first TaqMan (registered trademark) probe that recognizes a sequence that characterizes CYP2A6*1 in the region amplified by the primer set (b) and is labeled with a first fluorescent dye; (d) a second TaqMan probe that recognizes a sequence that characterizes CYP2A6*4 in the region amplified by the primer set (b) and is labeled with a second fluorescent dye different from the first fluorescent dye; performing amplification by the TaqMan method using the above; (3) measuring the fluorescence intensity caused by the first fluorescent dye and the fluorescence intensity caused by the second fluorescent dye after the completion of the step (2) and / or while the step (2) is being carried out; Including, The forward primer used in the step (1) is a primer that hybridizes to a common sequence of CYP2A6 and CYP2A7 located upstream on the 5' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs, and the reverse primer used in the step (1) is a primer that hybridizes to a CYP2A6-specific sequence located downstream on the 3' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs; The forward primer used in step (2) is a primer that hybridizes to a consensus sequence of CYP2A6 and CYP2A7 located upstream on the 5' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs, and the reverse primer used in step (2) is a primer that hybridizes to a consensus sequence of CYP2A6 and CYP2A7 located downstream on the 3' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs; The first TaqMan probe (c) used in step (2) is a probe that hybridizes to a CYP2A6-specific gene sequence that is not found in CYP2A7, located upstream on the 5' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs in the region amplified in the main amplification, and the second TaqMan probe (d) is a probe that hybridizes to a CYP2A7-specific gene sequence that is not found in CYP2A6, located upstream on the 5' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs in the region amplified in the main amplification; Each one uses When determining CYP2A6*7, (1) a step of performing preliminary amplification using a primer set that simultaneously amplifies the regions that characterize CYP2A6*1 and *7 but does not amplify the CYP2A7 gene; (2)(a) a template DNA obtained by diluting the amplification product obtained in the step (1); (b) a primer set capable of simultaneously amplifying a region of 200 bp or less containing the sequences characteristic of CYP2A6*1 and *7; (c) a first TaqMan (registered trademark) probe that recognizes a sequence that characterizes CYP2A6*1 in the region amplified by the primer set (b) and is labeled with a first fluorescent dye; (d) a second TaqMan probe that recognizes a sequence that characterizes CYP2A6*7 in the region amplified by the primer set (b) and is labeled with a second fluorescent dye different from the first fluorescent dye; performing amplification by the TaqMan method using the above; (3) measuring the fluorescence intensity caused by the first fluorescent dye and the fluorescence intensity caused by the second fluorescent dye after the completion of the step (2) and / or while the step (2) is being carried out; Including, The primer set used in the step (1) is a primer set that hybridizes to a CYP2A6-specific sequence and is capable of amplifying a region containing the amino acid at position 471 of CYP2A6; The primer set used in the step (2) is a primer set that hybridizes to a CYP2A6-specific sequence and is capable of amplifying a region containing the amino acid at position 471 of CYP2A6; The first TaqMan probe (c) used in step (2) is a probe that hybridizes to a region containing a sequence in which the amino acid at position 471 of CYP2A6 is isoleucine, and the second TaqMan probe (d) is a probe that hybridizes to a region (typically a region consisting of 10 to 50 bases, preferably a region consisting of 10 to 20 bases) containing a sequence in which the amino acid at position 471 of CYP2A6 is threonine; Each of the above methods is used. [2] When determining CYP2A6*4, The primer set used in the step (1) is a combination of a forward primer represented by a sequence consisting of 18 to 24 consecutive bases in the base sequence of SEQ ID NO: 12 and a reverse primer represented by a sequence consisting of 14 to 20 consecutive bases in the base sequence of SEQ ID NO: 13; The primer set (b) used in the step (2) is a combination of a forward primer represented by a sequence consisting of 21 to 27 consecutive bases in the nucleotide sequence of SEQ ID NO: 14 and a reverse primer represented by a sequence consisting of 21 to 27 consecutive bases in the nucleotide sequence of SEQ ID NO: 15; As the first TaqMan probe (c) and the second TaqMan probe (d) used in the step (2), a combination of a probe represented by a sequence consisting of 12 to 18 consecutive bases in the base sequence of SEQ ID NO: 16 and a probe represented by a sequence consisting of 12 to 18 consecutive bases in the base sequence of SEQ ID NO: 17 is used; Each one uses When determining CYP2A6*7, The primer set used in the step (1) is a combination of a forward primer represented by a sequence consisting of 18 to 24 consecutive bases in the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 18 and a reverse primer represented by a sequence consisting of 14 to 20 consecutive bases in the nucleotide sequence of SEQ ID NO: 13; The primer set (b) used in the step (2) is a combination of a forward primer represented by a sequence consisting of 18 to 24 consecutive bases in the nucleotide sequence of SEQ ID NO: 19 and a reverse primer represented by a sequence consisting of 16 to 22 consecutive bases in the nucleotide sequence of SEQ ID NO: 20; As the first TaqMan probe (c) and the second TaqMan probe (d) used in the step (2), a combination of a probe represented by a sequence consisting of 16 to 22 consecutive bases in the base sequence of SEQ ID NO: 21 and a probe represented by a sequence consisting of 16 to 22 consecutive bases in the base sequence of SEQ ID NO: 22 is used; Each uses the method in [1]. [3] When determining CYP2A6*4, The primer set used in the step (1) is a combination of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2; The primer set (b) used in the step (2) is a combination of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4; As the first TaqMan probe (c) and the second TaqMan probe (d) used in the step (2), a combination of a TaqMan probe consisting of the nucleotide sequence of SEQ ID NO: 5 and a TaqMan probe consisting of the nucleotide sequence of SEQ ID NO: 6 is used; Each one uses When determining CYP2A6*7, The primer set used in the step (1) includes a combination of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 7 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2; The primer set (b) used in the step (2) is a combination of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 8 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 9; a combination of a TaqMan probe consisting of the nucleotide sequence of SEQ ID NO: 10 and a TaqMan probe consisting of the nucleotide sequence of SEQ ID NO: 11 as the first TaqMan probe (c) and the second TaqMan probe (d) used in the step (2); Use method [1] or [2] respectively. [4] Any of the methods [1] to [3] for determining CYP2A6*4 and CYP2A6*7, wherein steps (2) to (3) for CYP2A6*4 and steps (2) to (3) for CYP2A6*7 are carried out in different reaction vessels. [5] A kit for determining CYP2A6*4, comprising a forward primer consisting of the base sequence of SEQ ID NO: 1, a reverse primer consisting of the base sequence of SEQ ID NO: 2, a forward primer consisting of the base sequence of SEQ ID NO: 3, a reverse primer consisting of the base sequence of SEQ ID NO: 4, a TaqMan probe consisting of the base sequence of SEQ ID NO: 5, and a TaqMan probe consisting of the base sequence of SEQ ID NO: 6. [6] A kit for determining CYP2A6*7, comprising a forward primer consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 7, a reverse primer consisting of the base sequence of SEQ ID NO: 2, a forward primer consisting of the base sequence of SEQ ID NO: 8, a reverse primer consisting of the base sequence of SEQ ID NO: 9, a TaqMan probe consisting of the base sequence of SEQ ID NO: 10, and a TaqMan probe consisting of the base sequence of SEQ ID NO: 11. [Effects of the Invention]

[0008] By using the determination method of the present invention, CYP2A6*4 and / or CYP2A6*7 (preferably CYP2A6*4 and CYP2A6*7) can be measured with high accuracy. Furthermore, since there is no need to detect fluorescence intensity in real time, it is possible to flexibly change the equipment used and the assay schedule. The present invention makes it possible to easily determine the majority of genotypes that cause PM of CYP2A6 in Asian populations, including Japanese. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a graph showing the results of clustering CYP2A6*4 by the method of the present invention. [Figure 2] 1 is a graph showing the results of clustering CYP2A6*7 by the method of the present invention. [Figure 3] 10 is a graph showing the results of clustering for CYP2A6*4 (no pretreatment), which is a comparative example. [Figure 4] 10 is a graph showing the results of clustering of CYP2A6*7 (no pretreatment), which is a comparative example. DETAILED DESCRIPTION OF THE INVENTION

[0010] In the method of the present invention, preliminary amplification (pre-amplification, pre-PCR) is performed, and then a portion of the amplified product is used for main amplification (hydrolysis probe method, TaqMan PCR). The subjects of determination in the method of the present invention are CYP2A6*1 (wild type), CYP2A6*4, and CYP2A6*7, which are genetic polymorphisms of CYP2A6, a type of cytochrome P450 (CYP) that catalyzes nicotine metabolism (Non-Patent Document 1). In the human genome, another gene (CYP2A7: SEQ ID NO: 24; translation start position is 38th base) with a base sequence very similar to that of CYP2A6 (SEQ ID NO: 23; translation start position is 22nd base) exists near the CYP2A6 locus, making it difficult to determine CYP2A6 genetic polymorphisms.

[0011] CYP2A6*1 is a wild-type mutant and exhibits normal nicotine metabolic activity. CYP2A6*4 lacks a large portion of the CYP2A6 gene as a result of non-homologous recombination between CYP2A6 and CYP2A7, resulting in a lack of enzyme activity. CYP2A6*7 is a single nucleotide polymorphism (SNP) in which isoleucine at position 471 is mutated to threonine, resulting in reduced enzyme activity (see Non-Patent Document 1). The method of the present invention requires an assay system that amplifies only the regions that characterize CYP2A6*1, CYP2A6*4, and CYP2A6*7, without amplifying another gene, CYP2A7, and that can clearly distinguish between them.

[0012] The sample used in the method of the present invention is not particularly limited as long as it contains genomic DNA. Examples include whole blood and cells. DNA can be obtained from these samples using conventional methods for DNA preparation.

[0013] (1) Determination of CYP2A6*4 As mentioned above, CYP2A6*4 is a non-functional allele that lacks most of the CYP2A6 gene due to non-homologous recombination between CYP2A6 and CYP2A7, and contains a fusion region of the 5' region of CYP2A7 and the 3' untranslated region of the CYP2A6 gene. Utilizing this, the region that characterizes CYP2A6*4, which lacks most of the gene, and the region that characterizes CYP2A6*1 are simultaneously amplified, and pre-amplified using primer sequences that do not amplify the separate gene CYP2A7.

[0014] Furthermore, using the amplified region as a template, hydrolysis probes that bind to the respective regions characteristic of CYP2A6*1 and CYP2A6*4 were designed, and the presence or absence of CYP2A6*1 and the presence or absence of CYP2A6*4 were converted into separate fluorescence values ​​by TaqMan PCR, allowing the determination of the CYP2A6 genotype (*1 / *1, *1 / *4, *4 / *4).

[0015] In the present invention, the "region characterizing CYP2A6*4" refers to a region that includes a site where non-homologous recombination between CYP2A6 and CYP2A7 occurs, and that contains a CYP2A6-specific sequence downstream on the 3' side and a CYP2A7-specific sequence upstream on the 5' side.

[0016] In the present invention, the "region characterizing CYP2A6*1" refers to a region that contains a site where non-homologous recombination between CYP2A6 and CYP2A7 occurs, and that contains a CYP2A6-specific sequence upstream of the 5' side of the site.

[0017] It has been reported that non-homologous recombination between CYP2A6 and CYP2A7 occurs frequently between 6657 bp to 6831 bp from the translation start site of the CYP2A6 gene sequence and 7049 bp to 7222 bp from the translation start site of the CYP2A7 gene sequence, or between 6750 bp to 6831 bp of the CYP2A6 gene sequence and 7141 bp to 7222 bp of the CYP2A7 gene sequence (FEBS Letters 448 (1999) 105-110).

[0018] In the present invention, a "primer set that simultaneously amplifies the regions characteristic of CYP2A6*1 and CYP2A6*4, but does not amplify the CYP2A7 gene" is a primer set that can simultaneously amplify unrecombined CYP2A6 and CYP2A6*4, which is the result of non-homologous recombination between CYP2A6 and CYP2A7, but does not amplify unrecombined CYP2A7.

[0019] By using a forward primer that hybridizes to a common sequence of CYP2A6 and CYP2A7 located upstream on the 5' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs, and a reverse primer that hybridizes to a CYP2A6-specific sequence located downstream on the 3' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs, CYP2A6*1 and CYP2A6*4 can be simultaneously amplified without amplifying CYP2A7. Preferably, the primers are designed so that the amplification products of the region characterizing CYP2A6*1 and the region characterizing CYP2A6*4 are approximately the same size.

[0020] CYP2A6*1 contains the allele CYP2A6*1B, in which a portion of the 3' untranslated region of the CYP2A6 gene is substituted with the CYP2A7 gene. Because the enzymatic activity of CYP2A6*1A (wild-type) and CYP2A6*1B is unchanged, the reverse primer must be designed to avoid the regions 6679 bp to 6737 bp and 7073 bp to 7099 bp from the translation start site of the CYP2A6 gene sequence where the substitution occurs, in order to distinguish between CYP2A6*1B and CYP2A6*4 in the method of the present invention. Because the phenotype (enzymatic activity) of CYP2A6*1A and CYP2A6*1B is unchanged, they are collectively referred to as CYP2A6*1 in this specification.

[0021] The length of each primer is not particularly limited as long as it is long enough to specifically recognize the target sequence in the human genome, but is generally in the range of 10 to 40 bases, preferably 15 to 30 bases, and particularly preferably 16 to 25 bases. The distance between primers, i.e., the amplified region, is not particularly limited as long as it is in the range of 100 to 5000 bases, but is preferably 100 to 2000 bases. The primers are designed so as to amplify a region including the base sequences of the primers and probe used in the main amplification.

[0022] The preliminary amplification in the method of the present invention can be carried out according to conventional methods, except for using the above-mentioned primers. However, by setting an appropriate extension time according to the length of the amplified region, it is possible to amplify only the region that characterizes each allele, without amplifying unrecombined CYP2A7.

[0023] In the method of the present invention, the amplification product obtained in the pre-amplification reaction is appropriately diluted and used as a template for the main amplification reaction. Generally, when performing two-step PCR, the obtained pre-amplification product is diluted 100 to 10,000 times with a nuclease-free solvent that does not inhibit the amplification in the main detection step, such as sterilized ultrapure water, and used as a template.

[0024] In the main amplification by the TaqMan method of the present invention, amplification is performed by the TaqMan method using a primer set that can simultaneously amplify a region of 200 bp or less containing the sequences characteristic of CYP2A6*1 and CYP2A6*4, and two probes labeled with different fluorescent dyes that recognize the sequences characteristic of CYP2A6*1 or CYP2A6*4 in the amplified region. TaqMan PCR generally uses labeled probes that anneal to part of the nucleotide sequence of the region to be amplified, and the target amplification product is detected by detecting the label released by the 5' exonuclease activity of the polymerase during the PCR extension reaction.

[0025] The primer set used for the main amplification can be set in the same way as the primer set used for pre-amplification, except that the distance between the primers is set to be approximately 200 bp or less, which is generally considered when using TaqMan probes, and that the primer set is set so that the region containing the base sequence of the probe can be amplified.

[0026] The sequence of the main amplification primer may partially overlap with or be identical to the sequence of the pre-amplification primer. For example, as shown in the Examples below, by using a primer set that hybridizes to the consensus sequence of CYP2A6 and CYP2A7, amplification can be achieved with one primer set whether the sequence 5' upstream of the site where nonhomologous recombination occurs is a CYP2A6-derived sequence (i.e., CYP2A6*1) or a CYP2A7-derived sequence (i.e., CYP2A6*4). More specifically, the forward primer used is a primer that hybridizes to the consensus sequence of CYP2A6 and CYP2A7 located 5' upstream of the site where nonhomologous recombination occurs between CYP2A6 and CYP2A7, and the reverse primer used is a primer that hybridizes to the consensus sequence of CYP2A6 and CYP2A7 located 3' downstream of the site where nonhomologous recombination occurs between CYP2A6 and CYP2A7.

[0027] A "probe that recognizes a sequence characteristic of CYP2A6*1" refers to a probe that hybridizes to a CYP2A6-specific gene sequence that is not found in CYP2A7, located upstream, 5' from the site where non-homologous recombination between CYP2A6 and CYP2A7 occurs, within the region amplified in the main amplification.

[0028] A "probe that recognizes a sequence characteristic of CYP2A6*4" refers to a probe that hybridizes to a gene sequence specific to CYP2A7 and not found in CYP2A6, located upstream, 5' from the site where non-homologous recombination between CYP2A6 and CYP2A7 occurs, within the region amplified in the main amplification.

[0029] The length of the probe is 10 to 50 bases, preferably 10 to 20 bases.

[0030] The probe used in the main amplification by the TaqMan method in the method of the present invention is labeled at one end with a fluorescent dye and at the other end with a quencher that inhibits the fluorescent dye.

[0031] In the present invention, a fluorescent dye refers to a substance that becomes excited by absorbing excitation light of a specific wavelength and emits fluorescence when returning to its original ground state. The fluorescent substance used for labeling is not particularly limited as long as it can label the end of the probe. For example, FAM, TET, HEX, Cy3, Cy5, Texas Red, TAMRA, FITC, etc. are suitable. Preferred examples include HEX (553 nm) and FAM (520 nm). In the method of the present invention, different fluorescent dyes are used in combination for the probe that recognizes the sequence characteristic of CYP2A6*1 and the probe that recognizes the sequence characteristic of CYP2A6*4. Fluorescent dyes with different detection wavelengths can be selected according to the analytical instrument used.

[0032] In the present invention, a quencher refers to a substance that absorbs the excitation energy of a fluorescent dye. Any of the following may be used: BHQ1, BHQ2, Dabcyl, etc. However, since the range of fluorescence suppression varies depending on the type of dye being suppressed, it is necessary to select a type of quencher that can suppress the emission of the fluorescent dye paired with it on a single probe. There are no particular restrictions as long as the range of emission suppression is within the range selected. For example, when the fluorescent dye is FAM or TET, BHQ1, which has a suppression range of wavelengths slightly toward the short wavelength side (480 nm to 580 nm), may be selected. When the fluorescent substance is Cy3 or Texas red, BHQ2, which has a suppression range of wavelengths slightly toward the long wavelength side (550 nm to 650 nm), may be selected.

[0033] In the present invention, "labeled" means that one of the fluorescent dye and the quencher is chemically bound to the 5'-end, and the other is chemically bound to the 3'-end. Usually, the fluorescent substance is bound to the 5'-end, but it does not matter if the fluorescent substance is bound to the 3'-end as long as the same effect is obtained.

[0034] The main amplification in the method of the present invention can be carried out according to the conventional TaqMan PCR method, except that a specific probe and primer pair are used. Those skilled in the art can optimize the PCR conditions as appropriate.

[0035] The determination method of the present invention utilizes the fluorescence emitted from the fluorescent dye. For example, in a probe whose 5' end is labeled with a fluorescent substance, a primer that hybridizes upstream of the probe and a DNA polymerase with 5' to 3' exonuclease activity are added to a gene amplification reaction solution, and gene amplification is carried out by PCR. The probe is degraded by the 5' to 3' exonuclease activity of the DNA polymerase, and the fluorescent dye is released from the probe, thereby releasing the inhibition by the quencher.

[0036] In the method of the present invention, CYP2A6*1 and CYP2A6*4 are detected by two colors of fluorescence intensity, respectively. This allows the use of general SNP determination software using hydrolysis probes. Visual determination is no longer necessary, eliminating the problems of misdetection and laborious work involved in conventional methods such as restriction enzyme digestion and electrophoresis. This allows for rapid determination of genetic polymorphisms in clinical settings and supports personalized drug formulation design. Furthermore, results can be more easily confirmed visually than by Tm value analysis.

[0037] The fluorescence intensity of the fluorescent dye can be measured in real time during the gene amplification reaction or after the reaction is complete. If measurement is performed during the amplification reaction, a dedicated real-time PCR device that combines a thermal cycler and a spectrofluorometer is required. If measurement is performed after the amplification reaction is complete, the gene amplification reaction can be performed using a thermal cycler, allowing for flexible selection of the device to be used depending on the scale of analysis.

[0038] In the method of the present invention, the primer sets and probes used for pre-amplification and main amplification may be designed according to methods known to those skilled in the art, taking into consideration the conditions used in PCR, such as ensuring uniform melting temperatures (Tm values), no intramolecular interactions, and avoiding complementary sequences that could form intermolecular dimers between the primers and probes. Probe and primer pairs may also be designed using appropriate primer-probe design software.

[0039] (2) Determination of CYP2A6*7 CYP2A6*7 is a single-base substitution allele (SNP ID: rs5031016). However, this SNP is located in a region highly homologous to CYP2A7 (position 6558 from the translation start site of the CYP2A6 gene), making it difficult to directly determine the genotype using a TaqMan probe. Therefore, for CYP2A6*7, we pre-amplified the region characterizing CYP2A6*7 and the region characterizing CYP2A6*1 simultaneously using primer sequences that do not amplify the separate gene CYP2A7. This results in specific amplification of only CYP2A6.

[0040] Furthermore, using the amplified region as a template, hydrolysis probes that bind to the same regions characteristic of CYP2A6*1 and CYP2A6*7 were designed, and the presence or absence of CYP2A6*1 and CYP2A6*7 were converted into separate fluorescence values ​​by TaqMan PCR, allowing the determination of the CYP2A6 genotype (*1 / *1, *1 / *7, *7 / *7).

[0041] In the present invention, the "region characterizing CYP2A6*1" and the "region characterizing CYP2A6*7" both refer to the region containing the amino acid at position 471 of CYP2A6. In the present invention, a "primer set that simultaneously amplifies regions characteristic of CYP2A6*1 and CYP2A6*7 but does not amplify the CYP2A7 gene" refers to a primer set that hybridizes to a CYP2A6-specific sequence and amplifies a region containing the amino acid at position 471 of CYP2A6. A "sequence characteristic of CYP2A6*1" refers to a sequence in which the amino acid at position 471 of CYP2A6 is isoleucine, and a "sequence characteristic of CYP2A6*7" refers to a sequence in which the amino acid at position 471 of CYP2A6 is threonine.

[0042] The primer set used for pre-amplification of CYP2A6*7 is a primer set that hybridizes to a CYP2A6-specific sequence and can amplify a region containing the amino acid at position 471 of CYP2A6. One or both of the forward and reverse primers may be identical to those of the primer set used for pre-amplification of CYP2A6*4, as long as they hybridize to a CYP2A6-specific sequence and can amplify a region containing the amino acid at position 471 of CYP2A6. By using the same primer set, pre-amplification for determining CYP2A6*4 and CYP2A6*7 can be performed in a single reaction vessel.

[0043] In the primary amplification of CYP2A6*7 using the TaqMan method, amplification is performed using a primer set that can simultaneously amplify a region of 200 bp or less containing the sequences characteristic of CYP2A6*1 and CYP2A6*7, and two types of probes labeled with different fluorescent dyes that recognize the sequences characteristic of CYP2A6*1 or CYP2A6*7 in the amplified region.

[0044] The primer set used for the main amplification is a primer set that hybridizes to a CYP2A6-specific sequence and is capable of amplifying a region of CYP2A6 containing amino acid 471. The sequence of the main amplification primer may partially overlap with or be identical to the sequence of the pre-amplification primer.

[0045] A "probe that recognizes a sequence characteristic of CYP2A6*1" refers to a probe that hybridizes to a region containing a sequence in which the amino acid at position 471 of CYP2A6 is isoleucine (typically a region consisting of 10 to 50 bases, preferably a region consisting of 10 to 20 bases).

[0046] A "probe that recognizes a sequence characteristic of CYP2A6*7" refers to a probe that hybridizes to a region containing a sequence in which the amino acid at position 471 of CYP2A6 is threonine (typically a region consisting of 10 to 50 bases, preferably a region consisting of 10 to 20 bases).

[0047] The requirements and principles other than the primers and probes used are the same as those for determining CYP2A6*4.

[0048] In the method of the present invention, in order to specifically detect CYP2A6*1, CYP2A6*4, and CYP2A6*7, the following primers and probes (1) to (3) can be designed and used to determine CYP2A6*4 (and CYP2A6*1), and the following primers and probes (4) to (6) can be designed and used to determine CYP2A6*7 (and CYP2A6*1). Primers and probes having complementary nucleotide sequences thereof may also be used.

[0049] (1) Preamplification primer sequence: A PCR primer pair that simultaneously amplifies the regions characteristic of CYP2A6*1 and CYP2A6*4 without amplifying the CYP2A7 gene, such as PCR-2A6st4-FW1 (SEQ ID NO: 1) and PCR-2A6-RW1 (SEQ ID NO: 2) listed in Table 1 below. The forward primer hybridizes to a consensus sequence of CYP2A6 and CYP2A7 located upstream on the 5' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs, and the reverse primer hybridizes to a CYP2A6-specific sequence located downstream on the 3' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs. Examples of such primer sets include a combination of a forward primer represented by a sequence consisting of 18 to 24 consecutive bases in the nucleotide sequence of SEQ ID NO: 12 and a reverse primer represented by a sequence consisting of 14 to 20 consecutive bases in the nucleotide sequence of SEQ ID NO: 13.

[0050] (2) Primary amplification primer sequence: A primer pair capable of simultaneously amplifying regions of 200 bp or less that characterize CYP2A6*1 and CYP2A6*4, such as Primer-2A6st4-FW1 (SEQ ID NO: 3) and Primer-2A6st4-RV1 (SEQ ID NO: 4) listed in Table 1. The forward primer hybridizes to a consensus sequence of CYP2A6 and CYP2A7 located upstream on the 5' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs, and the reverse primer hybridizes to a consensus sequence of CYP2A6 and CYP2A7 located downstream on the 3' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs. Examples of such primer sets include a combination of a forward primer represented by a sequence consisting of 21 to 27 consecutive bases in the nucleotide sequence of SEQ ID NO: 14 and a reverse primer represented by a sequence consisting of 21 to 27 consecutive bases in the nucleotide sequence of SEQ ID NO: 15.

[0051] (3) Hydrolysis probe sequence: a hydrolysis probe that recognizes each sequence characteristic of CYP2A6*1 and CYP2A6*4 in the region amplified by the primer pair (2), and that can be detected as separate fluorescence, as typified by Probe-2A6st4(HEX) (SEQ ID NO: 5) and Probe-2A7st4(FAM) (SEQ ID NO: 6) listed in Table 1. The probe that recognizes the sequence characteristic of CYP2A6*1 hybridizes to a CYP2A6-specific gene sequence that is not found in CYP2A7, located upstream on the 5' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs, within the region amplified by the main amplification, while the probe that recognizes the sequence characteristic of CYP2A6*4 hybridizes to a CYP2A7-specific gene sequence that is not found in CYP2A6, located upstream on the 5' side of the site where nonhomologous recombination between CYP2A6 and CYP2A7 occurs, within the region amplified by the main amplification. An example of a probe that recognizes a sequence characteristic of CYP2A6*1 is a probe represented by a sequence consisting of 12 to 18 consecutive bases in the base sequence of SEQ ID NO: 16. An example of a probe that recognizes a sequence characteristic of CYP2A6*4 is a probe represented by a sequence consisting of 12 to 18 consecutive bases in the base sequence of SEQ ID NO: 17.

[0052] (4) Preamplification primer sequence: A PCR primer pair that simultaneously amplifies the regions characteristic of CYP2A6*1 and CYP2A6*7 without amplifying the CYP2A7 gene, such as PCR-2A6st7-FW1 (SEQ ID NO: 7) [or PCR-2A6st4-FW1 (SEQ ID NO: 1)] and PCR-2A6-RW1 (SEQ ID NO: 2) listed in Table 3 below. This primer set hybridizes to a CYP2A6-specific sequence and can amplify a region containing the amino acid at position 471 of CYP2A6. Examples of such primer sets include a combination of a forward primer represented by a sequence consisting of 18 to 24 consecutive bases in the nucleotide sequence of SEQ ID NO: 12 or SEQ ID NO: 18 and a reverse primer represented by a sequence consisting of 14 to 20 consecutive bases in the nucleotide sequence of SEQ ID NO: 13.

[0053] (5) Primary amplification primer sequence: A primer pair capable of simultaneously amplifying regions of 200 bp or less that characterize CYP2A6*1 and CYP2A6*7, such as Primer-2A6st7-FW1 (SEQ ID NO: 8) and Primer-2A6st7-RV1 (SEQ ID NO: 9) listed in Table 3. This primer set hybridizes to a CYP2A6-specific sequence and amplifies a region containing the amino acid at position 471 of CYP2A6. Examples of such primer sets include a combination of a forward primer represented by a sequence consisting of 18 to 24 consecutive bases in the nucleotide sequence of SEQ ID NO: 19 and a reverse primer represented by a sequence consisting of 16 to 22 consecutive bases in the nucleotide sequence of SEQ ID NO: 20.

[0054] (6) Hydrolysis probe sequence: a hydrolysis probe that recognizes sequences characteristic of CYP2A6*1 and CYP2A6*7 in the region amplified with the primer pair (5), and that can be detected as different fluorescence, such as Probe-2A6st1(HEX) (SEQ ID NO: 10) and Probe-2A6st7(FAM) (SEQ ID NO: 11) shown in Table 3. The probe that recognizes the sequence characteristic of CYP2A6*1 hybridizes to a region (typically a region consisting of 10 to 50 bases, preferably a region consisting of 10 to 20 bases) containing a sequence in which the amino acid at position 471 of CYP2A6 is isoleucine, and the probe that recognizes the sequence characteristic of CYP2A6*7 hybridizes to a region (typically a region consisting of 10 to 50 bases, preferably a region consisting of 10 to 20 bases) containing a sequence in which the amino acid at position 471 of CYP2A6 is threonine. An example of a probe that recognizes a sequence characteristic of CYP2A6*1 is a probe represented by a sequence consisting of 16 to 22 consecutive bases in the base sequence of SEQ ID NO: 21. An example of a probe that recognizes a sequence characteristic of CYP2A6*7 is a probe represented by a sequence consisting of 16 to 22 consecutive bases in the base sequence of SEQ ID NO: 22.

[0055] The determination kit of the present invention is a kit that can be used in the method of the present invention, and is characterized by comprising a pre-amplification primer set, a main amplification primer set, and two types of probes. The probes and primer sets are as described above in relation to the method of the present invention. In the present determination kit, the probe and primer pair may be contained as a mixture or separately. The kit of the present invention may further contain reagents required for PCR in addition to the probe and primer set.

[0056] Pre-amplification and main amplification in the method of the present invention can be carried out according to conventional methods except for using the above-mentioned probes and primers, and more specifically, can be carried out, for example, according to the procedures described in the Examples below. [Example]

[0057] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0058] Example 1: Preparation of measurement samples, primers, and probes The genomic DNA used as the measurement sample was HapMap DNA (HAPMAP PT02) purchased from the Coriell Institute, diluted with commercially available diethylpyrocarbonate (DEPC)-treated water (Life Technologies Japan, Inc.) to a genomic DNA concentration of 2 ng / μL. The HapMap DNA used for the measurement is shown in Table 11.

[0059] To measure CYP2A6*4, the pre-PCR primers and TaqMan PCR primers and probes listed in Table 1 were prepared. The FW primer (PCR-2A6st4-FW1; SEQ ID NO: 1) hybridizes to the consensus sequence of CYP2A6 and CYP2A7, and the RV primer (PCR-2A6-RW1; SEQ ID NO: 2) hybridizes to the CYP2A6-specific sequence. This allows specific amplification of CYP2A6*1 and allele CYP2A6*4 without amplifying CYP2A7. The TaqMan PCR primers (Primer-2A6st4-FW1; SEQ ID NO: 3, Primer-2A6st4-RV1; SEQ ID NO: 4) hybridize to the consensus sequence of CYP2A6 and CYP2A7, allowing amplification of both CYP2A6*1 and CYP2A6*4. The probe for Allele X (Probe-2A6st4(HEX); sequence number 5) is a hydrolysis probe that recognizes a CYP2A6-specific sequence, i.e., a sequence that characterizes CYP2A6*1, in the TaqMan PCR amplification product, and the probe for Allele Y (Probe-2A7st4(FAM); sequence number 6) is a hydrolysis probe that recognizes a sequence that characterizes CYP2A7, i.e., CYP2A6*4, in the amplification product. The explanation (origin) of each sequence is shown in Table 2.

[0060] In addition, to measure CYP2A6*7, pre-PCR primers and TaqMan PCR primers and probes listed in Table 3 were prepared. Both the pre-PCR and TaqMan PCR primers can specifically amplify CYP2A6. The Allele X probe is a hydrolysis probe that recognizes a sequence characteristic of CYP2A6*1 in the amplified product, and the Allele Y probe is a hydrolysis probe that recognizes a sequence characteristic of CYP2A6*7 in the amplified product. Note that the bases indicated in lowercase letters in each base sequence of the TaqMan PCR probe indicate the position of the single-base substitution that characterizes CYP2A6*1 or CYP2A6*7. The explanation (origin) of each sequence is shown in Table 4.

[0061] [Table 1]

[0062] [Table 2]

[0063] [Table 3]

[0064] [Table 4]

[0065] Example 2: Pre-PCR Pre-PCR Primer Mix (2.5 μmol / L) for CYP2A6*4 and CYP2A6*7 measurements was prepared by mixing 2.5 μL each of the pre-PCR forward primer (100 μmol / L) and reverse primer (100 μmol / L) with 95 μL of DEPC-Treated Water. Next, according to the composition (for one reaction) listed in Table 5, the reagents included with the commercially available PCR reagent (TaKaRa LA Taq with GC Buffer; Takara Bio Inc.), the Pre-PCR Primer Mix (2.5 μmol / L), and DEPC-Treated Water were mixed and stirred, and then spun down to prepare the pre-PCR mixture. 12 μL of the pre-PCR mixture was dispensed into each well of a 96-well plate. Next, 3 μL of each HapMap DNA solution and 3 μL of DEPC-Treated Water (negative control) were added. The 96-well plate was sealed and mixed by tapping. After spinning down, the 96-well plate was set in a PCR system (GeneAmp PCR System 9700; Life Technologies Japan, Inc.) and reacted under the conditions shown in Table 6 to obtain a pre-PCR reaction product.

[0066] [Table 5]

[0067] [Table 6]

[0068] Example 3: TaqMan PCR TaqMan MGB Probe & Primer Mix (10x) was prepared according to the composition shown in Table 7. Next, the components were mixed in a test tube mixer according to the composition (for one reaction) shown in Table 8, and then spun down to prepare a PCR Reaction Mix. 6.0 μL of each PCR Reaction Mix was added to each well of a 384-well plate. Subsequently, 4.0 μL of a pre-PCR reaction product diluted solution prepared by diluting the pre-PCR reaction product obtained in Example 2 with DEPC-Treated Water 1024-fold and 4.0 μL of DEPC-Treated Water as a negative control were added. Reactions for each sample were performed in duplicate (two wells each). The 384-well plate was sealed, spun down, and then placed in a real-time PCR device (LightCycler 480 II; Roche Diagnostics).

[0069] [Table 7]

[0070] [Table 8]

[0071] Real-time PCR was carried out under the conditions shown in Table 9. After the reaction was completed, the fluorescence intensity at the endpoint was measured. The obtained measurement data was converted according to Table 10 to determine SNP. When measurement was impossible, N / A was displayed.

[0072] [Table 9]

[0073] [Table 10]

[0074] Example 4: Results and Discussion The results for CYP2A6*4 are shown in Table 11. The clustering results for CYP2A6*4 are shown in Figure 1. In Figure 1, the horizontal axis represents the fluorescence intensity of HEX (Allele X), and the vertical axis represents the fluorescence intensity of FAM (Allele Y). For all measured samples, duplicate results were identical and consistent with the prior information. Negative controls (no genomic DNA) were negative, with neither HEX nor FAM detected. Clustering clearly distinguished heterozygotes for allele X and allele Y (Both Alleles; upper right zone of the graph in Figure 1), homozygotes for allele X (Allele X; lower right zone of the graph in Figure 1), homozygotes for allele Y (Allele Y; upper left zone of the graph in Figure 1), and negative controls (Negative; lower left zone of the graph in Figure 1), enabling automatic classification. Two samples (NA18952 and NA18973) were classified as *4 / *4 (0 copies).

[0075] [Table 11]

[0076] The results for CYP2A6*7 are shown in Table 12. The clustering results for CYP2A6*7 are shown in Figure 2. In Table 12, "*Allele X" indicates a determination based on a low signal value. In Figure 2, the horizontal axis represents the fluorescence intensity of HEX (Allele X), and the vertical axis represents the fluorescence intensity of FAM (Allele Y). The results for NA18952 and NA18973 will be discussed later. Except for these, the duplicate results were identical for all other test samples. The negative control (without genomic DNA) showed no detection of either HEX or FAM, resulting in a negative result. Clustering clearly distinguished heterozygotes for Allele X and Allele Y (Both Alleles; upper right zone of the graph in Figure 2), homozygotes for Allele X (Allele X; lower right zone of the graph in Figure 2), homozygotes for Allele Y (Allele Y; upper left zone of the graph in Figure 2), and the negative control (Negative; lower left zone of the graph in Figure 2), enabling automatic identification. Sequencing of the pre-PCR reaction products confirmed that only the CYP2A6 gene had been amplified (i.e., the CYP2A7 gene was not amplified). The results for NA18952 and NA18973 were low signals, and considering that NA18952 and NA18973 were *4 / *4 (0 copies), this result was deemed reasonable.

[0077] [Table 12]

[0078] Comparison Example: TaqMan PCR (pre-PCR not performed) PCR reaction mixes were prepared as in Example 3, and 6.0 μL of each PCR reaction mix was added to each well of a 384-well plate. Subsequently, 4.0 μL of each HapMap DNA solution was added in place of the pre-PCR reaction product dilution solution, and DEPC-Treated Water was added as a negative control. Reactions were performed in duplicate (two wells per sample) for each sample. The 384-well plate was sealed and spun down, then placed in a real-time PCR instrument (LightCycler 480 II; Roche Diagnostics) and real-time PCR was performed under the conditions shown in Table 9.

[0079] The clustering results for CYP2A6*4 and CYP2A6*7 are shown in Figures 3 and 4, respectively. The horizontal axis represents the fluorescence intensity of HEX (Allele X), and the vertical axis represents the fluorescence intensity of FAM (Allele Y). In both figures, it was not possible to distinguish between heterozygotes for Allele X and Allele Y, homozygotes for Allele X, homozygotes for Allele Y, and the negative control. Generally, pre-PCR is not necessary for germ cell-derived gene polymorphism testing, as high-quality genomic DNA can be easily obtained from peripheral blood. However, it was found that pre-PCR is required for the measurement of CYP2A6*4 and CYP2A6*7.

[0080] Preparation Example: Primer and Probe Design Primers and probes that can be used in the method of the present invention are shown in Tables 13 and 14.

[0081] [Table 13]

[0082] [Table 14] [Industrial Applicability]

[0083] The present invention can be used to determine genetic polymorphisms of CYP2A6, and can be used for applications such as nicotine metabolism and the diagnosis, treatment, and prevention of diseases associated with nicotine metabolism.

Claims

1. A method for determining at least one of CYP2A6*4 and CYP2A6*7, which are alleles of CYP2A6, comprising: When determining CYP2A6*4, (1) a step of performing preliminary amplification using a primer set that simultaneously amplifies the regions that characterize CYP2A6*1 and CYP2A6*4 and does not amplify the CYP2A7 gene; (2) (a) a template DNA obtained by diluting the amplification product obtained in the step (1); (b) a primer set capable of simultaneously amplifying a region of 200 bp or less containing a sequence characteristic of CYP2A6*1 and CYP2A6*4; (c) a first TaqMan® probe that recognizes a sequence that characterizes CYP2A6*1 in the region amplified by the primer set (b) and is labeled with a first fluorescent dye; (d) a second TaqMan probe that recognizes a sequence that characterizes CYP2A6*4 in the region amplified by the primer set (b) and is labeled with a second fluorescent dye that is different from the first fluorescent dye; performing amplification by the TaqMan method using the following: (3) measuring the fluorescence intensity due to the first fluorescent dye and the fluorescence intensity due to the second fluorescent dye after the completion of the step (2) and / or while the step (2) is being carried out; Including, The primer set used in the step (1) is a combination of a forward primer represented by a sequence consisting of 18 to 24 consecutive bases in the base sequence of SEQ ID NO: 12 and a reverse primer represented by a sequence consisting of 14 to 20 consecutive bases in the base sequence of SEQ ID NO: 13; The primer set (b) used in the step (2) is a combination of a forward primer represented by a sequence consisting of 21 to 27 consecutive bases in the base sequence of SEQ ID NO: 14 and a reverse primer represented by a sequence consisting of 21 to 27 consecutive bases in the base sequence of SEQ ID NO: 15; As the first TaqMan probe (c) and the second TaqMan probe (d) used in the step (2), a combination of a probe represented by a sequence consisting of 12 to 18 consecutive bases in the base sequence of SEQ ID NO: 16 and a probe represented by a sequence consisting of 12 to 18 consecutive bases in the base sequence of SEQ ID NO: 17 is used; Each one uses When determining CYP2A6*7, (1) a step of performing preliminary amplification using a primer set that simultaneously amplifies the regions that characterize CYP2A6*1 and *7 and does not amplify the CYP2A7 gene; (2) (a) a template DNA obtained by diluting the amplification product obtained in the step (1); (b) a primer set capable of simultaneously amplifying a region of 200 bp or less containing a sequence characteristic of CYP2A6*1 and *7; (c) a first TaqMan® probe that recognizes a sequence that characterizes CYP2A6*1 in the region amplified by the primer set (b) and is labeled with a first fluorescent dye; (d) a second TaqMan probe that recognizes a sequence that characterizes CYP2A6*7 in the region amplified by the primer set (b) and is labeled with a second fluorescent dye that is different from the first fluorescent dye; performing amplification by the TaqMan method using the following: (3) measuring the fluorescence intensity due to the first fluorescent dye and the fluorescence intensity due to the second fluorescent dye after the completion of the step (2) and / or while the step (2) is being carried out; Including, The primer set used in the step (1) is a combination of a forward primer represented by a sequence consisting of 18 to 24 consecutive bases in the base sequence of SEQ ID NO: 12 or 18 and a reverse primer represented by a sequence consisting of 14 to 20 consecutive bases in the base sequence of SEQ ID NO: 13; The primer set (b) used in the step (2) is a combination of a forward primer represented by a sequence consisting of 18 to 24 consecutive bases in the base sequence of SEQ ID NO: 19 and a reverse primer represented by a sequence consisting of 16 to 22 consecutive bases in the base sequence of SEQ ID NO: 20; As the first TaqMan probe (c) and the second TaqMan probe (d) used in the step (2), a combination of a probe represented by a sequence consisting of 16 to 22 consecutive bases in the base sequence of SEQ ID NO: 21 and a probe represented by a sequence consisting of 16 to 22 consecutive bases in the base sequence of SEQ ID NO: 22 is used; Each of the above methods is used.

2. When determining CYP2A6*4, The primer set used in the step (1) is a combination of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2; The primer set (b) used in the step (2) is a combination of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 3 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 4; a combination of a TaqMan probe consisting of the base sequence of SEQ ID NO: 5 and a TaqMan probe consisting of the base sequence of SEQ ID NO: 6 as the first TaqMan probe (c) and the second TaqMan probe (d) used in the step (2); Each one uses When determining CYP2A6*7, The primer set used in the step (1) includes a combination of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 7 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2; The primer set (b) used in the step (2) is a combination of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 8 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 9; a combination of a TaqMan probe consisting of the base sequence of SEQ ID NO: 10 and a TaqMan probe consisting of the base sequence of SEQ ID NO: 11 as the first TaqMan probe (c) and the second TaqMan probe (d) used in the step (2); The method of claim 1, wherein each of the following is used:

3. 3. The method according to claim 1 or 2, for determining CYP2A6*4 and CYP2A6*7, wherein steps (2) to (3) for CYP2A6*4 and steps (2) to (3) for CYP2A6*7 are carried out in different reaction vessels, respectively.

4. A kit for determining CYP2A6*4, comprising a forward primer consisting of the base sequence of SEQ ID NO: 1, a reverse primer consisting of the base sequence of SEQ ID NO: 2, a forward primer consisting of the base sequence of SEQ ID NO: 3, a reverse primer consisting of the base sequence of SEQ ID NO: 4, a TaqMan probe consisting of the base sequence of SEQ ID NO: 5, and a TaqMan probe consisting of the base sequence of SEQ ID NO:

6.

5. A kit for determining CYP2A6*7, comprising a forward primer consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 7, a reverse primer consisting of the base sequence of SEQ ID NO: 2, a forward primer consisting of the base sequence of SEQ ID NO: 8, a reverse primer consisting of the base sequence of SEQ ID NO: 9, a TaqMan probe consisting of the base sequence of SEQ ID NO: 10, and a TaqMan probe consisting of the base sequence of SEQ ID NO: 11.

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