KR101072903B1 - htSNP FOR DETERMINING A GENOTYPE OF CYTOCHROME P450 2A6 GENE AND USE THEREOF - Google Patents

Abstract

The present invention relates to haplotype marker single nucleotide variation (htSNP) and its use for genotyping of cytochrome P450 2A6 gene, and more particularly, to a method for screening htSNP for haplotype analysis of human CYP2A6 gene and the htSNP. The present invention relates to a method for determining the functional variant of the CYP2A6 gene.

 By using the method of the present invention, htSNP obtained based on single nucleotide polymorphism (SNP) of Korean CYP2A6 gene was used for the CYP2A6 gene found in Asians with similar genetic characteristics to Koreans. Functional genotypes can be readily identified. In addition, it can be usefully used to predict individual differences in CYP2A6 enzymatic activity in these races or to predict abnormal symptoms caused by CYP2A6 deficiency.

CYP2A6, monobasic polymorphism, haplotype, htSNP

Description

Haplotype marker single base mutation and its use for genotyping of cytochrome P450 2A6 gene and its use {htSNP FOR DETERMINING A GENOTYPE OF CYTOCHROME P450 2A6 GENE AND USE THEREOF}

Figure 1 shows the type and frequency of haplotypes in Korean of CYP2A6 used to select htSNP combination in the present invention.

2 to 7 illustrate the htSNP combination selected in the present invention,

FIG. 2 illustrates htSNP combination selection for analyzing haplotypes of the CYP2A6 gene, including six functional mutations and three suspected functional genes in a genetic mutation test subject.

FIG. 3 illustrates htSNP combination selection to analyze haplotypes of the CYP2A6 gene, including eight amino acid substitution variants, three variants that mark deletions of the CYP2A6 gene, and six high frequency CYP2A6 gene variants.

FIG. 4 illustrates another htSNP combination selection for analyzing haplotypes of the CYP2A6 gene, including eight amino acid substitution variants, three variants labeling a deletion of the CYP2A6 gene, and six high frequency CYP2A6 gene variants.

FIG. 5 illustrates another htSNP combination selection for analyzing haplotypes of the CYP2A6 gene, including eight amino acid substitution variants, three mutations that label the deletion of the CYP2A6 gene, and six high frequency CYP2A6 gene variants.

FIG. 6 illustrates another htSNP combination selection for analyzing haplotypes of the CYP2A6 gene, including eight amino acid substitution variants, three variants that mark deletions of the CYP2A6 gene, and six high frequency CYP2A6 gene variants.

FIG. 7 illustrates yet another htSNP combination selection for analyzing haplotypes of the CYP2A6 gene, including eight amino acid substitution variants, three variants labeling a deletion of the CYP2A6 gene, and six high frequency CYP2A6 gene variants.

8 to 16 are snapshots of one type of htSNP combination and CYP2A6 functional mutagenesis combination selected in the present invention.

8 shows that the mutation positions of -48T> G, 2134A> G and 6558T> C of the CYP2A6 gene are heterozygous (Hetero) variants in which one strand of the two strands of DNA is mutant and the other strand has a wild type ego,

9 is a case where the 567C> T mutation position of the CYP2A6 gene is a heterozygous (Hetero) variant in which one strand of the two strands of DNA is a variant, and the other strand has a wild type,

10 is a case where the mutation position of 6458A> T and 6558T> C of the CYP2A6 gene is a heterozygous (Hetero) variant in which one strand of two strands of DNA is mutant and one strand is wild type,

FIG. 11 shows that the mutation positions of -48T> G, 13G> A and 6558T> C of the CYP2A6 gene are heterozygous (Hetero) mutations in which one strand of the two strands of DNA is mutant and the other strand has a wild type ego,

FIG. 12 shows a case where the mutation position of 3391T> C of the CYP2A6 gene is a heterozygous (Hetero) variant of one strand of the mutant and the other strand of the missing type;

FIG. 13 is a case where the mutation positions of -48T> G and 2134A> G of the CYP2A6 gene are one strand variant and the other strand is a heterozygous (Hetero) variant gene of the missing type;

14 shows that the mutation positions of -48T> G, 6558T> C, and 6600G> T of the CYP2A6 gene are heterozygous (Hetero) mutations in which one strand of the two strands of DNA is mutant and the other strand has a wild type Is the case,

FIG. 15 shows a case where the mutation position of 6458A> T of the CYP2A6 gene is a hetero variant of one strand, and the other strand is a heterozygous (Hetero) variant gene of a missing type,

FIG. 16 shows a case where the mutation position of 6558T> C and 6582G> T of the CYP2A6 gene is a heterozygous (Hetero) variant in which one strand of the two strands of DNA is mutant and the other strand has a wild type.

17 to 18 are examples of a snapshot method performed in parallel to the genetic test of FIGS. 8 to 16 to further examine a case where the CYP2A6 gene is deleted in addition to the gene mutation of FIGS. 8 to 16.

17 is a case where the CYP2A6 gene is normally present on the homologous chromosome,

18 illustrates a case in which the CYP2A6 gene has only one gene because it does not exist in one chromosome.

Figure 19 shows the conjugation form of the remaining portion of the CYP2A6 gene and CYP2A7 gene deletion of the CYP2A6 gene.

The present invention relates to htSNP for the genotyping of cytochrome P450 2A6 gene and its use, and more particularly to the selection method of htSNP for haplotype analysis of human CYP2A6 gene and to determine the genotype of CYP2A6 gene using the htSNP. It is about how to.

Genetic diversity between individuals results in differences between individuals in drug toxicity and efficacy. Therefore, considering the effects on the genetic diversity of such pharmaceutically important proteins in the early stages of drug development is an important factor that can reduce the risk of drug development failure. It is the "haplotype" to be one research group that measures the genetic diversity between these individuals. Haplotypes refer to the combination of polymorphisms in each gene sequence present in one population, which provides more accurate and reliable information about genetic diversity than individual polymorphisms.

Human cytochrome P450, on the other hand, is a member of heme proteins that promote the oxidation of foreign chemicals such as drugs, carcinogens and toxins and internal substrates such as steroids, fatty acids and vitamins (Nelson et al ., Pharmacogenetics 6: 1-42 , 1996). Various subtypes of cytochrome P450 have been found in organs such as the liver, kidneys, intestines and lungs. Among them, the human cytochrome P450 2A6 (Cytochrome P450 2A6, hereinafter referred to as 'CYP2A6') gene is located on the 19th chromosome, and upstream of the CYP2A6 gene, CYP2A7, a pseudogene with high sequence similarity, is located.

CYP2A6 enzyme is an important enzyme that is responsible for about 80% of nicotine metabolism by breaking down nicotine into cotinine and 5-fluorouracil, which is an active form of the anticancer drug tegafur in vivo. It is an enzyme involved in the process of conversion to (5-fluorouracil, 5-FU). The enzyme is produced mainly in the liver and is expressed in small amounts in organs such as lung, colon, breast, kidney and uterus ( Drug Metab Dispos ., 29 (2): 91-5, 2001; Adv Drug Deliv Rev. , 18; 54 (10): 1245-56, 2002).

The activity of the enzymes in the individual is so diverse that they are classified into groups of pore metabolizers (PM), intermediate metabolizers (IM), extensible metabolizers (EM) and ultrarapid metaboilzers (UM), respectively. The different activity of enzymes as described above may be caused by genetic polymorphism of CYP2A6 gene. As of June 2007, more than 50 genotypes of CYP2A6 are known (http://www.cypalleles.ki.se/cyp2a6.htm), and the frequency and type of genotypes show distinct differences among species. However, since various types of gene mutations and haplotypes have been reported, it is important to determine the correct haplotype through minimal nucleotide polymorphism (SNP) in order to increase analysis time and cost efficiency. Do.

The haplotype tagging SNP (htSNP) screening technique is a method for increasing the efficiency of diagnosis. The htSNP screening technique is a method for selecting a minimum number of mutant gene combinations for accurate display of each haplotype, and all haplotypes can be predicted if only the selected SNP is identified.

In many genes, the distribution of genetic polymorphisms is known to be different according to the species. In the case of the CYP2A6 gene, there are no unique genetic variants and haplotypes that occur frequently in Koreans, and if so, how often It is necessary to check how the selection of htSNP is performed. However, studies on the genotype of CYP2A6 gene and htSNP for analyzing it in Korean have been insufficient.

Therefore, the present inventors have been studying to develop a method that can accurately identify mutations in the CYP2A6 gene mainly found in Korean in a short time, select htSNP for the mutation of the CYP2A6 gene mainly found in Korean to determine the CYP2A6 gene The present invention was completed by confirming that the method of determining the genotype has excellent efficiency such as time and cost.

Accordingly, it is an object of the present invention to provide a method for selecting htSNPs capable of analyzing haplotypes of the CYP2A6 gene found in Koreans.

In addition, another object of the present invention to provide a method for determining the genotype of the human CYP2A6 gene using the htSNP.

To achieve these and other advantages and in accordance with the purpose of the present invention,

1) taking a biological sample from the subject;

2) extracting nucleic acids from the sample taken in step 1);

3) performing PCR with a primer capable of amplifying the human CYP2A6 gene or fragment thereof as a template using the nucleic acid of step 2);

4) confirming the presence or absence of a mutation in the base sequence of the PCR product obtained in step 3);

5) analyzing haplotypes from the nucleotide sequences of PCR products identified as having a mutation in step 4); And

6) provides a method for screening the htSNP of the human CYP2A6 gene comprising the step of selecting the htSNP by analyzing the nucleotide sequence of the haplotype analyzed in step 5) using SNPtagger software.

In order to achieve the other object of the present invention,

1) taking a biological sample from the subject;

2) extracting nucleic acids from the sample taken in step 1);

3) performing PCR with a primer capable of amplifying the human CYP2A6 gene or fragment thereof as a template using the nucleic acid of step 2); And

4) -48T> G, 13G> A, 567C> T, 2134A> G, 3391T> C, 6458A> T, 6558T> C, 6582G> T, 6600G> T in the nucleotide sequence of the PCR product obtained in step 3) And it provides a method for determining the genotype of the human CYP2A6 gene comprising the step of examining the presence or absence of CYP2A6 gene mutation selected from the group consisting of 6091C> T.

In the present invention, a "biological sample" includes blood, skin cells, mucosal cells and hair of a subject, and preferably may be blood.

In the present invention, the nucleic acid may be DNA or RNA, preferably DNA, more preferably genomic DNA.

In the present invention, the term "aN> M" or "NaM" (where a is an integer and N and M are each independently A, C, T or G) means that the ath N base in the gene sequence is the M base. It means substituted by. For example, the "-48T> G" side refers to the substitution of the -48th base from T to G in the nucleotide sequence of the human CYP2A6 gene.

Hereinafter, the present invention will be described in detail.

The present invention is to identify the genotype of the CYP2A6 gene mainly found in Korean through mutation analysis of the CYP2A6 gene in Korea, based on the selection of htSNP, the optimal label set of each haplotype, and confirmed its usefulness There is this.

The method for selecting htSNPs of the human CYP2A6 gene according to the present invention includes the following steps:

1) taking a biological sample from the subject;

2) extracting nucleic acids from the sample taken in step 1);

3) performing PCR with a primer capable of amplifying the human CYP2A6 gene or fragment thereof as a template using the nucleic acid of step 2);

4) confirming the presence or absence of a mutation in the base sequence of the PCR product obtained in step 3);

5) analyzing haplotypes from the nucleotide sequences of PCR products identified as having a mutation in step 4); And

6) selecting htSNP by analyzing the nucleotide sequence of the haplotype analyzed in step 5) using SNP tagger software (http://www.well.ox.ac.uk/~xiayi/haplotype/).

The method of extracting nucleic acids from the sample collected in step 1) is not particularly limited, and techniques known in the art or commercially available extraction kits may be used. For example, kits for DNA or RNA extraction can be purchased from Qiagen (USA) and Stratagene (USA). In the case of extracting and using RNA from the above, cDNA is prepared by reverse transcription.

In step 3), the fragment of the human CYP2A6 gene refers to a fragment containing a known variation of the human CYP2A6 gene, such as single nucleotide polymorphism (SNP). Primers capable of amplifying the human CYP2A6 gene or fragments thereof may be designed based on the nucleotide sequence of the human CYP2A6 gene or fragments thereof, for example, may be selected from the group consisting of the primers of SEQ ID NOs: 2-15. However, the present invention is not limited thereto.

The mutations identified in step 4) include, but are not limited to, SNPs, deletion of genes and duplication of genes, and may include, for example, 30 variations shown in Table 4.

In addition, the presence or absence of the mutation in step 4) may be performed by mutation detection methods known in the art, and preferably, wild type CYP2A6 gene known in the art using sequencing and electrophoresis analysis. Base sequence of, for example, the base sequence of SEQ ID NO: 1 (GenBank accession No .: NC_000019), or by comparing based on each base sequence of CYP2A6 genotypes known in the art, or wild type using RFLP analysis or the like This can be done by comparing the restriction enzyme cleavage pattern of the CYP2A6 gene. In addition, in the case of deletion or duplication of the CYP2A6 gene can be confirmed by electrophoretic analysis of the PCR product. The sequencing can be performed using an autobase sequencer or using pyrosequencing.

Analysis of haplotypes from nucleotide sequences of PCR products identified as having mutations in step 5) can be predicted using SNPAlyze, Haplotyper, or Arlequin.

The method of the present invention may further comprise repeating steps 1) to 5). If you want to analyze the mutation patterns of the CYP2A6 gene in a certain population such as race or patient, select the CYP2A6 genotypes found in the population by investigating the frequency of each CYP2A6 genotype. Can be done.

In step 6), the nucleotide sequence of the haplotype analyzed in step 5) is analyzed by SNP tagging software to select htSNP, and the software used for selection of htSNP is HapBlock, LDSelect, Haploview, htSNP, TagIT, tagSNPs in addition to SNPtagger. Etc. can be used. The SNPtagger software is known in the art, and preferably http://www.well.ox.ac.uk/~xiayi/haplotype/. The selected htSNPs can be verified to increase the accuracy in diplotype determination. The verification can be performed using Matlab (The Math Works Inc., USA).

In one embodiment of the present invention, in order to select htSNP for the CYP2A6 genotype of Koreans, the mutation of the CYP2A6 gene found in Koreans was first examined. As a result, a total of 30 SNPs were found in Korean CYP2A6 gene (see Table 4).

In another embodiment of the present invention using the SNPAlyze program of DYNACOM for 14 SNPs including 8 mutations and 6 frequent mutations resulting in amino acid substitutions including functional gene mutations among the 30 SNPs selected above Haplotypes were analyzed to identify haplotypes with a frequency of more than 19%. The program used for haplotype analysis is not limited to the SNPAlyze program, Haplotyper (http://www.people.fas.harvard.edu/~junliu/Haplo/docMain.htm), Arlequin (http: // Many similar softwares are known in the art, such as lgb.unife.ch/arlequin) and SNP Analyzer from Istec (http://www.istech21.com/).

In another embodiment of the present invention, 20 haplotypes, including the 19 haplotypes and genetic defects, are selected to select htSNP, which is a minimal marker that can easily distinguish the genotype of the CYP2A6 gene, which is mainly found in Koreans. The nucleotide sequence and frequency for htSNP were selected by analysis using SNP tagger software, examples of which are shown in FIGS. 2 to 7.

The htSNP combination selected in the present invention by the above method can be used to analyze the genotype of the human CYP2A6 gene. Thus, the present invention provides a method for determining the genotype of the human CYP2A6 gene. The method includes the following steps:

1) taking a biological sample from the subject;

2) extracting nucleic acids from the sample taken in step 1);

3) performing PCR with a primer capable of amplifying the human CYP2A6 gene or fragment thereof as a template using the nucleic acid of step 2); And

4) -48T> G, 13G> A, 567C> T, 2134A> G, 3391T> C, 6458A> T, 6558T> C, 6582G> T, 6600G> T in the nucleotide sequence of the PCR product obtained in step 3) And the presence or absence of a CYP2A6 gene mutation selected from the group consisting of 6091C> T.

Extracting the nucleic acid in the step 2) is as described above.

The fragment of the human CYP2A6 gene refers to a fragment containing a known variant of the human CYP2A6 gene, such as SNP, as described above. Primers that can be used in step 3) may be, but are not limited to, primers of SEQ ID NOs: 16 and 17.

The mutation irradiated in step 4) may be selected from htSNPs shown in FIGS. 2 to 7. For example, in step 4), -48T> G shown in FIG. 2; 22C> T; 567C> T; 2134A> G; 3391T> C; 6458A> T; 6558T> C; 6582G> T; 6600G> T; One in the group consisting of 6091C> T, 5971G> A and 5983T> G; 13G> A; 51G> A; 1620T> C; And 1836G> T; You can investigate the presence of a mutation consisting of,

-48T> G shown in FIG. 3; 22C> T; 51G> A; 567C> T; 1620T> C; 1836G> T; 3391T> C; 6458A> T; 6558T> C; 6600G> T; In the group consisting of 6091C> T, 5971G> A and 5983T> G, the presence of a single mutation can be investigated.

22C> T shown in FIG. 4; 51G> A; 567C> T; 1620T> C; 1836G> T; 3391T> C; 6354T> C; 6458A> T; 6558T> C; 6600G> T; In the group consisting of 6091C> T, 5971G> A and 5983T> G, the presence of a single mutation can be investigated.

-48T> G shown in FIG. 5; 13G> A; 22C> T; 51G> A; 567C> T; 1620T> C; 1836G> T; 2134A> G; 3391T> C; 6458A> T; 6558T> C; In the group consisting of 6091C> T, 5971G> A and 5983T> G, the presence of a single mutation can be investigated.

-48T> G shown in FIG. 6; 13G> A; 22C> T; 51G> A; 567C> T; 1620T> C; 1836G> T; 3391T> C; 6458A> T; 6558T> C; In the group consisting of 6091C> T, 5971G> A and 5983T> G, the presence of a single mutation can be investigated.

-48T> G shown in FIG. 7; 22C> T; 51G> A; 567C> T; 1620T> C; 1836G> T; 2134A> G; 3391T> C; 6458A> T; 6558T> C; 6600G> T; In the group consisting of 6091C> T, 5971G> A, and 5983T> G, the presence or absence of one mutation may be examined, and preferably, the presence or absence of the mutation illustrated in FIG. 2 may be investigated.

Among the mutations illustrated in FIG. 2, the most probable or functionally expected mutations are mutations that replace amino acids or deletions of genes, and therefore, the detection of functional CYP2A6 mutations is most preferred. 2, amino acid substitution mutations and gene deletion mutations can be examined. In the case of gene defects, it is difficult to discriminate by single nucleotide polymorphism. Therefore, we need to find a mutation that can label gene defects. The 6091C> T mutation is a single nucleotide polymorphism specifically found on a chromosome in which the CYP2A6 gene is deleted in the PCR product amplified in step 3), and may be used as a marker mutation for gene deletion. The combination of mutations selected for this purpose of discrimination is -48T> G; 13G> A; 567C> T; 2134A> G; 3391T> C; 6458A> T; 6558T> C; 6582G> T; 6600G> T; And 10 variations of 6091C> T. Instead of the 6091C> T variant, other variants that label gene deletions may use 5971G> A and 5983T> G instead of the CYP2A6 gene.

Since the mutation examined in step 4) is for the CYP2A6 gene mutation mainly found in Koreans, it is very specific for determining the haplotype and genotype of the CYP2A6 gene in Koreans.

In step 4), whether the mutation of the CYP2A6 gene is present in the base sequence of the PCR product may be performed using a polymorphism analysis method known in the art. Snapshot analysis (see Peter M. Vallone, et al., Int J Legal Med , 2004, 118: 147-157), electrophoretic analysis or combinations thereof, more preferably snapshot analysis. Can be.

The snapshot analysis is a method of genotyping through a PCR reaction using a ddNTP and a primer having a sequence annealed to the adjacent region of the SNP (not including the SNP region). Snapshot analysis used in the present invention can be designed and manufactured according to a known method based on the SNP of the CYP2A6 gene to be investigated in the step 3), the base immediately next to the SNP position is 3 'end and It includes a sequence that is annealed to the adjacent region of the SNP position, and 5 'terminal can be used without limitation if the T base is added, preferably a primer selected from the group consisting of the primers of SEQ ID NO: 23 to 28 can be used . At this time, the sequence annealed to the adjacent region of the SNP position is preferably about 20 bp in length, if you want to distinguish several SNPs at once the length of the 5 'terminal T base of the snapshot primers for each SNP Designed differently, for example, by adding 5 more T base at the 5 'position to make a difference in size between the primers can be synthesized by varying the length of the PCR product. The ddNTP complementary to each SNP is coupled to the snapshot primers thus produced, and these compounds can be distinguished from several SNPs at a time because the length difference occurs according to the SNPs.

Thereafter, the base sequence of the PCR product amplified for snapshot analysis can be analyzed by sequencing methods known in the art, and is not particularly limited, but preferably may be automatic base sequence analysis.

For example, when the htSNP combination shown in FIG. 2 in step 3), a primer having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 26 may be used, and preferably SEQ ID NO: 18 to SEQ ID NO: All 26 primers may be used, but the present invention is not limited thereto.

Thereafter, the nucleotide sequence of the PCR product amplified by the snapshot analysis may be analyzed by sequencing methods known in the art, but is not particularly limited, but may preferably be an automatic sequencing method.

In another embodiment of the present invention confirmed the usefulness of the htSNP combination selected in the present invention. To this end, in order to perform a snapshot analysis by selecting 10 functional or functional predictive CYP2A6 mutations among the htSNP combinations shown in FIG. 2, the base sequence of the PCR product obtained in step 3 was analyzed. As a result, it was confirmed that the method of the present invention can simultaneously and rapidly analyze the CYP2A6 genotype found in Koreans (see FIGS. 8 to 18).

Genotypes of the CYP2A6 gene that can be analyzed by the methods of the present invention are -48T> G, 13G> A, 567C> T, 2134A> G, 3391T> C, 6458A> T, 6558T> C, 6582G> T, 6600G> T, and 6091C> T, and are shown in FIGS. 8-18 for each genotype and resulting variation. For example, referring to FIG. 8, the genotype has mutations at three positions of -48T> G, 6558T> C, and 2134A> G, and indicates the wild type at the remaining seven positions. 9 is a genotype with mutations only at the 567C> T position and wild type at the remaining nine positions. In addition, the CYP2A6 * 4 genotype is a genotype including a 2A6 deletion mutation, and the CYP2A6 gene is deleted on the human chromosome so that no enzyme production occurs. When the CYP2A6 gene is deleted, the shape of the gene has a shape in which a part of the CYP2A6 gene and a part of the CYP2A7 are linked to each other, and thus, the mutation-specific mutation can be found by examining this part.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1  : Genotyping of 2A6 Gene in Koreans

<1-1> Amplification of 2A6 Gene

After separating blood from 50 healthy subjects, each DNA was isolated using Qiagen's genomic DNA separation kit. The CYP2A6 gene is about 6.9 kb in length, including nine exons. Therefore, PCR was performed by dividing the CYP2A6 gene into seven fragments. Primers used for each PCR are shown in Table 1 below. In the base sequences described in this specification, A, T, G, and C mean adenine, thymine, guanine, and cytosine, respectively.

 Primer for CYP2A6 Gene Amplification and Its Sequence PCR products Fryer Name Sequence (5 '→ 3') SEQ ID NO: CYP2A6_exon1 exon1F * GGTCTTCCTCCCCTTCCCAAT 2 exon1.R * CCCAAGATCCTGTCTTTCT 3 CYP2A6_exon2 exon2F TGTGTCCCAAGCTAGGCAGG 4 exon2R GGGAAGACCAGACTGGGGAC 5 CYP2A6_exon3,4 exon3,4F * CTCTGACTGAGTTTGCAGCTCTG 6 exon3,4R * GGGACACTGTCTGGAGGGC 7 CYP2A6_exon5 exon5F * GCCCCACTGAAATACCTAAACAAC 8 exon5R * GGGCCTGTGTCATCTGCCT 9 CYP2A6_exon6 exon6F * CCCTCTTTCCACCTTTGGTCTGA 10 exon6R * AAGTCACGTCTCAGGGTCCC 11 CYP2A6_exon7,8 exon7,8F * GCTCTGAGACCCCTAGATACC 12 exon7,8R * GCCCCTGCTGGTGTGAGCC 13 CYP2A6_exon9 exon9F * GCAAGTGTACCTGGCAGGAAA 14 exon9R * TGTAAAATGGGCATGAACGCCC 15 *: Drug Metab . Pharmacokin , 17 (5): SNP18 (482) -SNP23 (487) (2002).

The location of each primer and the size of PCR products are shown in Table 2 below. In addition, the location of the nucleotides was described according to the nomenclature of the Cytochrome P450 (CYP) Allele Nomenclature Committee (http://www.cypalleles.ki.se/cyp2a6.htm).

 Position of primer and size of PCR product PCR products Primer Name SEQ ID NO: location Size of PCR Product CYP2A6_exon1 exon1F 2 ? 764 ～ 744 1088 bp exon1.R 3 306 ～ 324 CYP2A6_exon2 exon2F 4 ? 151 ～ 132 884 bp exon2R 5 713 ～ 732 CYP2A6_exon3 ~ 4 exon3,4F 6 1504-1526 812 bp exon3,4R 7 2297 ～ 2315 CYP2A6_exon5 exon5F 8 3238 ～ 3261 402 bp exon5R 9 3621 ～ 3639 CYP2A6_exon6 exon6F 10 4217 ～ 4239 364 bp exon6R 11 4561 ～ 4580 CYP2A6_exon7,8 exon7,8F 12 4899-4919 947 bp exon7,8R 13 5827 ～ 5845 CYP2A6_exon9 exon9F 14 6082 ～ 6102 898 bp exon9R 15 6958 ～ 6979

Reaction conditions for each PCR fragment are shown in Table 3 below.

 PCR reaction conditions PCR products Reaction conditions CYP2A6_exon1 94 ° C 5 minutes, (94 ° C 30 seconds, 63 ° C 30 seconds, 72 ° C 65 seconds) 35 cycles, 72 ° C 5 minutes CYP2A6_exon2 94 ° C 4 minutes, (94 ° C 30 seconds, 63 ° C 30 seconds, 72 ° C 50 seconds) 35 cycles, 72 ° C 5 minutes CYP2A6_exon3,4 94 ° C 4 minutes, (94 ° C 30 seconds, 60 ° C 30 seconds, 72 ° C 40 seconds) 35 cycles, 72 ° C 5 minutes CYP2A6_exon5 94 ° C 4 minutes, (94 ° C 30 seconds, 63 ° C 30 seconds, 72 ° C 40 seconds) 35 cycles, 72 ° C 5 minutes CYP2A6_exon6 94 ° C 4 minutes, (94 ° C 30 seconds, 63 ° C 30 seconds, 72 ° C 40 seconds) 35 cycles, 72 ° C 5 minutes CYP2A6_exon7,8 94 ° C 5 minutes, (94 ° C 30 seconds, 66 ° C 30 seconds, 72 ° C 60 seconds) 35 cycles, 72 ° C 5 minutes CYP2A6_exon9 94 ° C 4 minutes, (94 ° C 30 seconds, 66 ° C 30 seconds, 72 ° C 60 seconds) 35 cycles, 72 ° C 5 minutes

<1-2> Sequence analysis of PCR product

The base sequence of each PCR product obtained in Example <1-1> was analyzed using an automatic sequence analyzer and primers of SEQ ID NOs: 2 to 15.

Then, when compared with the nucleotide sequence of the wild-type CYP2A6 gene (SEQ ID NO: 1), a total of 27 SNPs were found, of which two were new SNPs that have not been reported to date. In addition, three SNPs were discovered through structural analysis by gene deletion, and a total of 30 SNPs are as shown in Table 4 below.

 Mutations in the CY2A6 Gene Found in Koreans SNP Allele location Amino acid mutations frequency (%) -495A> G # Promoter 1.19 -48T> G Promoter 28.57 13G> A Exon 1 G5R 1.19 22C> T Exon 1 L8L 25.00 51G> A * 1B12 Exon 1 V17V 14.29 144G> A Exon 1 Q48Q 1.19 237G> A Intron 1.19 411C> T Intron 1.19 567C> T Exon 2 R101X 1.19 1620T> C Intron 84.52 1836G> T Intron 15.48 1890G> C Intron 1.19 2134A> G Exon 4 K194E 2.38 3391T> C * 11 Exon 5 S224P 1.19 3492C> T Exon 5 R257R 1.19 3570C> G Intron 1.19 5336G> A Intron 1.19 5628C> T Intron 2.38 5636A> C Intron 2.38 6218A> G Intron 1.19 6282A> G Intron 2.38 6293T> C Intron 2.38 6354T> C Intron 30.95 6458A> T # Exon 9 N438Y 3.57 6558T> C * 7 Exon 9 I471T 20.23 6582G> T * 5 Exon 9 G479V 1.19 6600G> T *8 Exon 9 R485L 5.95 5971G> A? *4 gene deletion 16.00  5983T> G? *4 gene deletion 16.00 6091C> T? *4 gene deletion 16.00

In the above table, " ⁺ " indicates that the CYP2A6 gene is deleted and a part of the remaining CYP2A6 gene and the CYP2A7 gene are combined (Fig. 19, all of the CYP2A6 genes 1 to 8 exons are lost, and the CYP2A6 terminal at the end of the exon 9 portion of CYP2A7 gene). As the mutation found in the gene of exon No. 9 of which part is partially substituted, the number is used according to the sequence assuming that the CYP2A6 gene is intact (since it is a deficiency of the CYP2A6 gene, it is called the SNP of the CYP2A6 gene). Difficult to see).

To distinguish the haplotypes that were deleted using SNPs in this structure, a forward primer at the 5 'position was designed within the same region of CYP2A6 and CYP2A7, and specific to CYP2A6 and the CYP2A7 gene was not amplified in the exon 9 region. By designing reverse primers, the pure CYP2A7 gene can not be amplified, but the intact CYP2A6 gene or CYP2A6 gene can be deleted to amplify a portion of the gene conjugated with the CYP2A7 gene.

In the amplified PCR product, CYP2A6 and CYP2A7-specific bases are selected. Based on ATG, the CYP2A6 translation initiation codon, the example of 6091C> T base of CYP2A6 (SEQ ID NO: 1) is shown below the CYP2A7 6091C region as shown below. Number: 30) It is similar to the base sequence of 6521T. It can be seen that these sites differ from CYP2A7 in 5971G and 5983T in CYP2A6 sequence as well as 6091.

In addition, in the above table, "*" means an allelic trait recognized by the International CYP Naming Commission, for example, * 11 is a variation in which the 224th amino acid is changed from serine to proline as compared to the wild type. The haplotype type has an international nomenclature that follows http://www.cypalleles.ki.se/cyp2a6.htm.

In addition, "#" in the table means a new variation.

Example 2: Haplotype Analysis of Genotypes of CYP2A6 Gene

Mutations of the 27 CYP2A6 genes and three CYP2A6 deletion marker variants identified in Example 1 are likely to affect the enzyme activity of CYP2A6 depending on the combination. Therefore, the present inventors analyzed the haplotype due to the mutations identified in Example 1 using the SNPAlyze program of DYNACOM. In general, in the analysis of haplotypes, it is difficult to secure statistical significance in the case of low frequency mutations, and thus, the distribution of haplotypes is mainly predicted by selecting mutations having a frequency of 5% or 10% or more. However, in the case of causing amino acid substitution, despite the low frequency, it has distinct functionalities, so in the present invention, a high frequency variation of -48T> G, 22C> T, 51G> A, 1620T> C, 1836G> T 8, 13G> A, 567C> T, 2134A> G, 3391T> C, 6458A> T, 6558T> C, 6582G> T, 6600G> T, resulting in 6 mutations and 6354T> C changes Haplotype analysis was performed using the mutations, and the mutations that could be labeled if there were gene defects were added to the test mutations, 5971G> A, 5983T> G, 6091C> T. As a result of analyzing haplotypes using a total of 17 mutations, a haplotype distribution was obtained in a total of 20 Koreans as shown in FIG. 1.

Example 3: Screening and Validation of htSNPs

Haplotypes, which are combinations of SNPs of the CYP2A6 gene, have been reported in several haplotypes for the potential to affect the enzyme activity of CYP2A6. This detailed haplotype information can be identified with minimal markers. These minimum markers are called htSNPs, which are necessary markers for the accurate representation of each haplotype and constitute various combinations. In order to select the htSNP combination, which is the optimized label set, a total of 20 haplotype sequences selected in Example 2 were selected from SNP tagger software (http://www.well.ox.ac.uk/~xiayi/). haplotype /) was used for analysis.

As a result, htSNP combinations as shown in Figures 2 to 7 were selected. Each of the selected htSNP combinations, shown in FIGS. 2-7, is an optimal label set, with '1' representing wild type, '2' representing variant, and 'V' designating each selected htSNP.

Once the htSNP analysis determines the genotype of each variant, the diplotype can be predicted from the results. However, since combinations of different haplotypes may have the same genotype, Matlab software (version 7.1, The Math Works) can determine whether a diplotype genotype can be determined without considering overlapping diplotypes among htSNP combinations. Inc., USA).

As a result of the test, it was confirmed that only the htSNP determined by this example can determine the ebe type without overlapping each other. This indicates that the htSNP combinations selected in the present invention are not identical to each other and thus there is no uncertain analysis in determining the genotype.

Example  4: Detection of Functional Variation of Fast CYP2A6 Gene

Among the 27 variants of the CYP2A6 gene found in the Korean identified in Example 1 and the three variants labeling the CYP2A6 gene deficiency, the genotype of CYP2A6, which changes its functionality, may be usefully used for gene diagnosis. Thus, -48T> G, 13G> A, 567C> T, 2134A> G, 3391T> C, 6458A> T, 6558T> C, which caused changes in the amino acids among the variations in FIG. Snapshot analysis, one of the fastest genotyping techniques of the CYP2A6 gene, for the rapid detection of a total of 10 functional variants, including 9 variants of 6582G> T, 6600G> T and 6091C> T mutations that mark gene defects Was carried out. In the selected htSNP, 10 variants reflecting the functionality of the htSNP combination of FIG. 2 are selected, and the positions of the variants are shown in Table 5 below.

Location of mutations screened with htSNP combinations according to the present invention Corresponding variations Mutation position htSNP 1 SNP1 -48T> G htSNP 2 SNP 2 13G> A htSNP 3 SNP 5 567C> T htSNP 4 SNP 8 2134A> G htSNP 5 SNP 9 3391T> C htSNP 6 SNP 11 6458A> T htSNP 7 SNP 12 6558T> C htSNP 8 SNP 13 6582G> T htSNP 9 SNP 14 6600G> T htSNP 10 SNP 15 6091C> T

Specifically, PCR was performed using the subject's DNA as a template and the amplified product was analyzed by snapshot. At this time, the primers used for PCR are shown in Table 6 below.

At this time, since the primer for amplifying the CYP2A6_long is a primer for amplifying the full-length gene of CYP2A6, it cannot be applied in the case of the CYP2A6 gene defect, and it is possible to amplify the CYP2A6 * 4 product to determine 6091C> T that marks the gene defect. CYP2A6 delF and CYP2A6 delR primer pairs should be used.

 Primer name and sequence PCR products Fryer Name Sequence (5 '→ 3') SEQ ID NO: CYP2A6_long CYP2A6 longF CTCTCCCCTGGAACCCCCAG 16 CYP2A6 longR GCACTTATGTTTTGTGAGACATCAGAGACAA 17 CYP2A6 * 4 CYP2A6 delF AGAATCTACCCTTGAGCCAGCA 28 CYP2A6 delR TGTAAAATGGGCATGAACGCCC 29

Reaction conditions for the PCR product are shown in Table 7 below.

 PCR reaction conditions PCR products Reaction conditions CYP2A6_long 94 ° C 1 minute, (98 ° C 20 seconds, 62 ° C 30 seconds, 72 ° C 7 minutes 30 seconds) 30 cycles, 72 ° C 10 minutes CYP2A6 * 4 94 ° C 5 minutes, (94 ° C 30 seconds, 58 ° C 30 seconds, 72 ° C 1 minute 20 seconds) 35 cycles, 72 ° C 7 minutes

Since unreacted primers and dNTPs of the amplified PCR products as described above may affect the snapshot process, to remove them, add 2 µl of ExoSAP-IT (USB) per 5 µl of the mixed PCR products. The reaction was carried out at 37 ° C. for 30 minutes and then at 80 ° C. for 15 minutes to deactivate the remaining enzyme. The enzyme-treated product was subjected to PCR reaction by making multiplex snapshot (multiplex SNaPshot) reaction using the primers shown in Table 9 below. Multiplexed snapshot reactants and PCR reaction conditions are shown in Tables 9 and 10, respectively.

 Primer and its base sequence Fryer Name Sequence (5 '→ 3') SEQ ID NO: Mu_-48T> G GGCTGGGGTGGTTTGCCTTT 18 Mu_13G> A_F TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCTACCACCATGCTGGCCTCA 19 Mu_567C> T_R TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAAGGTGGCTTGCTCGCCTC 20 Mu_2134A / G_R TTTTTTGACAGGAACTCTTTGTCCT 21 Mu_3391T / C_F TTTTTTTTTTCCCAGCTCTATGAGATGTTC 22 Mu_6458A> T_R TTTTTTTTTTTTTTTCAGGCCTTCTCCGAAACAGT 23 Mu_6558T / C_F TTTTTTTTTTTTTTTTTTTTCTCCCAGTCACCTAAGGACA 24 Mu_6582G> T_F TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTGTCCCCCAAACACGTGG 25 Mu_6600G / T_R TTTTTTTTTTTTTTTTTTTTTTTTTGGAAGCTCATGGTGTAGTTT 26 Multi2A6 / 7_6091C> T_F CAGTCATATTTGCAAGTGT 27

 Multiplex Snapshot Reactant  Furtherance Volume (μl / sample) Snapshot Multiplex Ready Reaction Mix
(SNaPshot Multiplex Ready Reaction Mix, ABI) 2 1/2 term buffer? 3 Enzymatic PCR Product 3 primer Primer Name density Mu_-48T> G 20 pM 0.1 Mu_13G> A_F 20 pM 0.1 Mu_567C> T_R 20 pM 0.1 Mu_2134A / G_R 20 pM 0.1 Mu_3391T / C_F 20 pM 0.1 Mu_6458A> T_R 20 pM 0.1 Mu_6558T / C_F 20 pM 0.1 Mu_6582G> T_F 20 pM 0.1 Mu_6600G / T_R 20 pM 0.1 Distilled water 1.4 Sum 10

(" ⁺ " Composition of 1/2 Term Buffer: 200 mM Tris-HCl, 5 mM MgCl ₂ , pH9; Nucleic Acids Research, 30 (15): 74, 2002)

PCR products Reaction conditions Snapshot product 40 cycles (96 ° C 10 seconds, 50 ° C 5 seconds, 60 ° C 30 seconds)

After the reaction was completed, 1 μl of SAP (USB) was added to 10 μl of the snapshot product to remove the remaining ddNTP, and reacted at 37 ° C. for 60 minutes and 65 ° C. for 15 minutes. At the end of the reaction, 0.5 μl of the reaction, 9.3 μl of Hi-Di formamide (ABI) and 0.2 μl of GeneScan-LIZ size standard (ABI) were mixed and denatured at 95 ° C. for 5 minutes. Then, it was analyzed by a 3100 gene analyzer (3100 Genetic Analyzer, ABI), the results are shown in Figures 8 to 16, the genotype analysis for this is shown in Table 11 below.

As shown in Figures 8 to 16, the color and size of the peaks were consistent with each SNP, and the color and position of the peaks were displayed differently according to the functional variant of the CYP2A6 gene. Heterozygous) and variants (isotypes) with homozygous alleles can be easily identified. In the graphs of FIGS. 8 to 16, the X axis represents the distance traveled by the primers in the autobase sequence analyzer by the difference in length of each primer, and the Y axis represents the intensity of fluorescence emitted from the fluorescent material having a specific wavelength incorporated in each base.

17 to 18 are examples of a snapshot method performed in parallel to the genetic test of FIGS. 8 to 16 to further examine a case where the CYP2A6 gene is deleted in addition to the gene mutation of FIGS. 8 to 16. Is a case where the CYP2A6 gene is normally present on the homologous chromosome, and FIG. 18 is a case where the CYP2A6 gene has only one gene because it is not present on one chromosome.

50 samples were analyzed by the snapshot method developed through the above invention, and full-length sequences were analyzed for the same sample, and the genotype readings were 100% identical. This indicates that the method of the invention is highly reproducible and accurate.

Therefore, it can be seen that the functional variants of the CYP2A6 gene can be analyzed easily and in a time and cost manner through the analysis method according to the present invention.

Therefore, this method is capable of analyzing the CYP2A6 genotypes by these combinations at the same time by distinguishing 10 CYP2A6 haplotypes mainly found in Koreans. Is high. In addition, almost all genotypes can be analyzed in the case of Japanese who have very similar genetic characteristics to Koreans, and it is considered that the CYP2A6 genotype can be determined in the range of 90% or more from Chinese known results.

As described above, htSNP for genotyping of the CYP2A6 gene according to the present invention and a method for analyzing functional variants of the CYP2A6 gene using the same are time and cost using htSNP obtained based on the SNP of the CYP2A6 gene in Korea. Efficiently identifying functional variants of the CYP2A6 gene found in Asians with similar genetic characteristics to Koreans. In addition, it can be usefully used to predict individual differences in CYP2A6 enzyme activity of Asians and abnormal signs that may be caused by CYP2A6 deficiency.

<110> INJE UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> htSNP FOR DETERMINING A GENOTYPE OF CYTOCHROME P450 2A6 GENE AND          USE THEREOF <130> DPP070115KR <160> 30 <170> KopatentIn 1.71 <210> 1 <211> 6897 <212> DNA <213> Homo sapiens <400> 1 actaccacca tgctggcctc agggatgctt ctggtggcct tgctggtctg cctgactgta 60 atggtcttga tgtctgtttg gcagcagagg aagagcaagg ggaagctgcc tccgggaccc 120 accccattgc ccttcattgg aaactacctg cagctgaaca cagagcagat gtacaactcc 180 ctcatgaagg tgtcccaagg cagggagatg ggtggcacgg ggtgggggct gcctagttgg 240 ctggggcttt gtggcagggg gttgaccagt gtggaccaga gtcttaggaa atggagtttt 300 ggagtttcag catcagaaag acaggatctt gggatgtcca gctccctgac tgtgagaacc 360 tgggtgcgaa gcatcccagc acatgacatc tcggtgctgg gccccattca gagtggaggc 420 ttctccctct aaccactccc acccacctcc atcagatcag tgagcgctat ggccccgtgt 480 tcaccattca cttggggccc cggcgggtcg tggtgctgtg tggacatgat gccgtcaggg 540 aggctctggt ggaccaggct gaggagttca gcgggcgagg cgagcaagcc accttcgact 600 gggtcttcaa aggctatggt gagggggtgc ccaagagggg gaaggtggcc aggtggacac 660 gaaggtctca gtgttcccag ccttctccct gactctcctg ccaactggag gctaaggcag 720 agtccccagt ctggtcttcc ctccccatct cccttcattg tggcctctcc atgtgtatcc 780 ctcacctgtc tccagcgtcc ctgtcctgat tcctccctgc ctctctctgc cccacctcct 840 tattctctct cactggagtc tcctctttcc cctctctctc catctctaag gacatcctgg 900 gtttctgttt tccagccctg gttctctgtc ttcatttgtc tttttgtccc tcttagcttc 960 tgggcttctc tgtgtttctc atctctccgc atccctttct cacttcttcc tctgtcttag 1020 gatttcaggg tattcctact tccacatctt cagcctccat ctcctggtaa cagtctctct 1080 tccttccaga ccctctctgt ttctatctca atatttttct ctcttctcca gctcagctta 1140 agaatctttc accattttta tttcctcctc ccagatctcc ccatatctca cttcccctcc 1200 ctccatcctc ttcctccatc actctctttc tctccccact tccttccctt cctccatgga 1260 gtgtcccctt atccctctgt ttctatgtgc atctctctgt ctggcctttc tgtttctttt 1320 ctgattgtct tattctttaa acctggtctc tctctctctc tctctctctc tctctctctc 1380 tctctctctc ctctctctct tctctctctc tctcctctct ctcttctctc tctctctctc 1440 tctctctcct ctctctcttc tctctctctc tctctctctc tctctctctc tctctacctc 1500 gacatcgtgt ttctctgact gagtttgcag ctctgctggg caatggcgct gaatccatct 1560 ctctcccacc ctactccctc tctcctccac ccttggggag ccccttggag ctgctccgct 1620 cctgcccctg ccgccccctg gcctgtctcc attcccgcgt tcacctcccc aggcgtggta 1680 ttcagcaacg gggagcgcgc caagcagctc cggcgcttct ccatcgccac cctgcgggac 1740 ttcggggtgg gcaagcgagg catcgaggag cgcatccagg aggaggcggg cttcctcatc 1800 gacgccctcc ggggcactgg cggtgagcag gggaccccga gtgcgggggc aggagaagga 1860 aaacacccag gacgaggaac ccgcgcgcgt tctgcctggg gatggggact aggtggggaa 1920 aggcgcccgc acttccagcc ctggagtctg gcgctgggaa tttggctcaa caaggccctg 1980 cctcctggaa ttctgactct cctcagacct ctgagttgac tctctcccca acccccttct 2040 cccgccatac ccgcaggcgc caatatcgat cccaccttct tcctgagccg cacagtctcc 2100 aatgtcatca gctccattgt ctttggggac cgctttgact ataaggacaa agagttcctg 2160 tcactgttgc gcatgatgct aggaatcttc cagttcacgt caacctccac ggggcaggta 2220 actggctgca gcccggcccg tgacgcccct accacaacct gccaactgct cccctacctg 2280 gagacaggtg ccccaaactc ccaccgccct ccagacagtg tcccctcaaa atcagtcccc 2340 gatattggac aactggacag ttgcaccaga acccaggatg gatgtccaat accctgtctc 2400 caaggacacc tggatagctc aacagatgct ccccaaaaca gagcctgctg gcaggatgca 2460 taccctcagc tcagctctct cacctgggca cgtgttccca tccccaactt accgtaattt 2520 ctaacagatg ctccctaccc agttcttctg aatattttaa cacctggaca agtgactgcg 2580 tcaaccggcc tcctgcatac ctgaacacct ggtgctgcaa aatccagccc atcataatca 2640 tttcacatct acacaaatgt cccagattag cccactggaa tatctggcca agcgccctct 2700 acttcaccca cttaaatacc tgaaaacatg gacagctgcc ctaaccaaca ccttaaacac 2760 ataaatatct agacagattg ttccctgaca cacaaataag tgttccccaa ccctttccaa 2820 tcacacacct tacagaggtg ctcccagtgc atcccacttg gataggtaaa cacctcaaca 2880 ggtatcccct ccacttcaac atcttcacca gccccacttt aatacctgag cacctgaaca 2940 aaagccccca atccagaccc agtaagtatc tggacagctg tctccaacca agcctacttg 3000 aatgcctaaa tacctagaca ggtgacactc acctcatacc agccccacct gaagagctaa 3060 acacctggac aggtgtcttc caactcaact tcacttgaat atctgaacac ctagatgtgt 3120 gctccaatcc agcctcgttt aaatacctga aacctggata tatgtctcag ttcttctcac 3180 ctaaattact agaccgtggc cctggtacct aaccttcctg aaaacttaga tataagttcc 3240 tatccggccc cactgaaata cctaaacaac gagacagatg cctttaactc agttccttcc 3300 ttgctatgaa acaaatccca ttcccatcag ctcctgcccc gtgacagctg tccttccctt 3360 cccatcctct ctctgcaacc ccagctctat gagatgttct cttcggtgat gaaacacctg 3420 ccaggaccac agcaacaggc ctttcagttg ctgcaagggc tggaggactt catagccaag 3480 aaggtggagc acaaccagcg cacgctggat cccaattccc cacgggactt cattgactcc 3540 tttctcatcc gcatgcagga ggtacacccc agcagccact gcggggagat gcaaagccag 3600 gcagagggaa atcagtctgg gagtggggca ggcagatgac acaggcccat tcaaattaac 3660 cctcatcata ataatcctca caattggctg ggtgccgtgg ctaacagcct gtaatcccag 3720 cactttggga ggccgaggca ggtggatcac ctgaggtcag gagttcgaga ccagcctggc 3780 caacatggtc aaaccccgtc tctactaaaa atccaaaaat tagttgggca tggtggcgcg 3840 aaggggggca gaggttgcaa tgagccaaga tcacggcatt gcactccagt ctgggtgaca 3900 gaatgaggcc ctgtgtcaaa aaaaattaat taattgttta aaaagtaagt gagcctgcat 3960 ggtcatgcgc atgtgcagtt ccagctactc aggaggctga ggctggagga ttgcttgagc 4020 tcaggagttg gagtccggcc tgtgcaactt agcaagacca agtcagtata agaaaaaaaa 4080 aaacaaaaaa aaactgacag ctaagttgat aattgaagga cagatggtca gcaaggtaaa 4140 gaaggtgaga aggaagagca ttttgggcaa agccagcagc cagggcaagg gctggaacct 4200 agagcgagtc tggtagatct agggtccctc tttccacctt tggtctgaac caaagagagg 4260 tagatccaaa ggaaaagccc tagaagggcc ctgagggcaa gaggagtgag gttggcctaa 4320 agccccctct ccctgcagga ggagaagaac cccaacacgg agttctactt gaaaaacctg 4380 gtgatgacca cgttgaacct cttcattggg ggcaccgaga ccgtcagcac caccctgcgc 4440 tatggcttct tgctgctcat gaagcaccca gaggtggagg gtaaggctgg agggggacgg 4500 aagtggaggg ccccagaccc tcaaaattcc ccttcgactg gtgcaatgtc cccacctgtc 4560 ccagatcccg ggaccctgag acgtgacttg ctgtccagag acagggcaac attcagctgg 4620 taggcatcag ctgagtctca ttagatatta aaatattgaa aatgtctgca ctgattggtc 4680 agtcacttct gtcccaagcc cactgagtgc ccactgcccg ttccaccggg tcatccccta 4740 agttcctccc tgtgcctccc ctgtgattct ggcacaacct ggttaacagg atcctactcc 4800 aacaatgcga atggctgatg tctgttctgt tatgaatgct ctacttccgt ctcataggcg 4860 gagccatatc atccacccca ttttgcctat tcggactatc atttcctgct ctgagacccc 4920 tagataccta aacacattcc ccctcctccc ccagccaagg tccatgagga gattgacaga 4980 gtgatcggca agaaccggca gcccaagttt gaggaccggg ccaagatgcc ctacatggag 5040 gcagtgatcc acgagatcca aagatttgga gacgtgatcc ccatgagttt ggcccgcaga 5100 gtcaaaaagg acaccaagtt tcgggatttc ttcctcccta aggtgctatc cgcccccacc 5160 ccccagacta cggggacttc cagcccctct ctgtgtcccc agcatcccac ccacattaga 5220 agctttctag accctgtccc actccctcaa tcagtcaaaa aagacttccc caaccaccac 5280 atccgttcca cctttccact tagacactcc tgagtcctgc atctctccag actctttgtg 5340 tcaggagaat caaacacatg ttcccaaact tcctatctta agaaacagaa gccccctttc 5400 cattcggcct tttgtcatag ggacagaaat ctcaggtccc ccaaactcct gcctagaagg 5460 acatggaccc catgtctccc aaacttcctg tttcagagat gtgaaccttc tatcccccaa 5520 agctcctcca tcagaggacc ccaactcctc catgcctgcc acttcccctt acctggggca 5580 cactagttcc ccctccagcc cctgtgtact ttcaccaatc ccccgaccct cctcatcaca 5640 cacaacttcc tcctccctac cagggcaccg aagtgttccc tatgctgggc tctgtgctga 5700 gagaccccag tttcttctcc aacccccagg acttcaatcc ccagcacttc ctgaatgaga 5760 aggggcagtt taagaagagt gatgcttttg tgcccttttc catcggtaag agaccactgt 5820 ttgctgccag gccacggctc acaccagcag gggcctccct caccctcctc ccctctctgc 5880 ggtgtagcct ggtatttctc cagcttggaa gttcctgtta gaatctaccc ttgagccagc 5940 agctgatact tccttaacta ccaagcaccc agtacctgcg cccaggtaaa atgaaaggaa 6000 acatctttcc ccgtagatgt atttctctag ggtcacacag cagattcctc agatccctaa 6060 aaaggagatg acggcacagc agtcatattt gcaagtgtac ctggcaggaa aggacatcta 6120 aacctcccat tgctacacct ggcatggatc accccatcta tgatggatgt gtgacattat 6180 gcctttttca aaacccatag aactgtataa cacagagtaa accctaatgt aaactatgga 6240 ctttgttagt aataatatat caatattggt tcaccattgt tacatctctt atagaaagaa 6300 attgaggctc agggaggatc agagcctcct ctgaaactct ctcaggccat aatattccac 6360 ccttcctccc tgggagagcc gcagctggag gtcggtactg gggcgaggct gcactgagag 6420 tgggcttcac ctccacccct cccgcctctc ctcctcagga aagcggaact gtttcggaga 6480 aggcctggcc agaatggagc tctttctctt cttcaccacc gtcatgcaga acttccgcct 6540 caagtcctcc cagtcaccta aggacattga cgtgtccccc aaacacgtgg gctttgccac 6600 gatcccacga aactacacca tgagcttcct gccccgctga gcgagggctg tgccggtgca 6660 ggtctggtgg gcggggccag ggaaagggca gggccaagac cgggcttggg agaggggcgc 6720 agctaagact gggggcagga tggcggaaag gaaggggcgt ggtggctaga gggaagagaa 6780 gaaacagaag cggctcagtt caccttgata aggtgcttcc gagctgggat gagaggaagg 6840 aaacccttac attatgctat gaagagtagt aataatagca gctcttattt cctgagc 6897 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> exon1F <400> 2 ggtcttcctc cccttcccaa t 21 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> exon1.R <400> 3 cccaagatcc tgtctttct 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> exon2F <400> 4 tgtgtcccaa gctaggcagg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> exon2R <400> 5 gggaagacca gactggggac 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> exon 3,4F <400> 6 ctctgactga gtttgcagct ctg 23 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> exon 3,4R <400> 7 gggacactgt ctggagggc 19 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> exon5F <400> 8 gccccactga aatacctaaa caac 24 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> exon5R <400> 9 gggcctgtgt catctgcct 19 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> exon6F <400> 10 ccctctttcc acctttggtc tga 23 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> exon6R <400> 11 aagtcacgtc tcagggtccc 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> exon 7,8 F <400> 12 gctctgagac ccctagatac c 21 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> exon 7,8 R <400> 13 gcccctgctg gtgtgagcc 19 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> exon9F <400> 14 gcaagtgtac ctggcaggaa a 21 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> exon9R <400> 15 tgtaaaatgg gcatgaacgc cc 22 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CYP2A6 longF <400> 16 ctctcccctg gaacccccag 20 <210> 17 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> CYP2A6 longR <400> 17 gcacttatgt tttgtgagac atcagagaca a 31 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Mu_-48T> G <400> 18 ggctggggtg gtttgccttt 20 <210> 19 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> Mu_13G> A_F <400> 19 tttttttttt tttttttttt tttttttttt ttttttttct accaccatgc tggcctca 58 <210> 20 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Mu_567C> T_R <400> 20 tttttttttt tttttttttt tttttttttt tttttttttt tttgaaggtg gcttgctcgc 60 ctc 63 <210> 21 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Mu_2134A / G_R <400> 21 ttttttgaca ggaactcttt gtcct 25 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Mu_3391T / C_F <400> 22 tttttttttt cccagctcta tgagatgttc 30 <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Mu_6458A> T_R <400> 23 tttttttttt tttttcaggc cttctccgaa acagt 35 <210> 24 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Mu_6558T / C_F <400> 24 tttttttttt tttttttttt ctcccagtca cctaaggaca 40 <210> 25 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Mu_6582G> T_F <400> 25 tttttttttt tttttttttt tttttttttt ttttcgtgtc ccccaaacac gtgg 54 <210> 26 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Mu_6600G / T_R <400> 26 tttttttttt tttttttttt tttttggaag ctcatggtgt agttt 45 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Multi2A6 / 7_6091C> T_F <400> 27 cagtcatatt tgcaagtgt 19 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CYP2A6 delF <400> 28 agaatctacc cttgagccag ca 22 <210> 29 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CYP2A6 delR <400> 29 tgtaaaatgg gcatgaacgc cc 22 <210> 30 <211> 7314 <212> DNA <213> Homo sapiens <400> 30 ccctgggtct tcctagcccc gagactttca agtccatatg cctggagtcc cccctcctga 60 gacccttaac cctgcatcct ccgcaacaga agacctccag atgcacagcc acacttccat 120 ctcaccctaa taaaacccag acctttggat tcctctccct tggaatgccc aaatccacaa 180 ctttggggtg cattctcact ctcagacccc aaatccaaag cccaagtgct cccctatgca 240 aatattccaa actcttcagt tctacagttt atcggttgcc ccctcctaaa tccacagccc 300 tgcggcaccc ctcctgaagt accacagatt tagtctggag gccccctctc tgttcagctg 360 ccctggggtc cccttatcct cccttgctgg ctgtgtccca agctaggtgg cattcatggt 420 ggggcgtgta gttgggaggt gaaataaggt gattatgtaa ttagccaaag tccatccctc 480 tttttcaggc agtataaagg caaaccaccc cacccatcac catctgtcat ctcactacca 540 ccatgctggc ctcagggctg cttctggtgg ccttgctggc ctgcctgact gtgatggtct 600 tgatgtctgt ctggcagcag aggaagagca gggggaagct gcctccggga cccaccccac 660 tgcccttcat tggaaactac ctccagctga acacagagca catatgtgac tccatcatga 720 aggtgtccca aggcaggaag atgggtggca cagggtgggg gctgcctagt tggctggggc 780 tttgtggcag gggattgacc agtgtggacc agagtcttag gaaagggagt tttggagttt 840 cagcatccgg gtcctagcca ggaaagacag gatcttggga tgtccagctc cctgactgtg 900 agaacctggg ggtgaaggat cccagtactt gacatctcgg tgctgggccc cattcagagt 960 gggggctgct ccctctaacc actcccactc gcctccatca gttcagtgag tgctatggcc 1020 ccgtgttcac cattcacttg gggccccggc gggtcgtggt gctgtgtgga catgatgccg 1080 tcagggaggc tctggtggac caggctgagg agttcagcgg gcgaggcgag caagccacct 1140 tcgactgggt cttcaaaggc tatggtgagg gggtgcccaa gatggggaag gtggccaggc 1200 ggacacgatg gtctcagtgt tgccagcctt ctccctgact ctcctgccca ctggaggcta 1260 tggcagaacc cccggtctgg tcttatctcc ccatctccct tcactgtggc ctctccatgt 1320 gtatccctca cctgtctcca gcgtccctgt cgtgattcct ccctgcctct ctctgcccca 1380 cctccttatt ctctctcact ggagtctcct ctttcccctc tctctccatc tctgaggaca 1440 tcctgggttt ctgttttcca gccctggtcc tctgtcttca tttgtctttt tgttgctctc 1500 agcttctgtg cttctccatg tttctcctct ctccacttcc ctctctcact ttttcctctc 1560 ccttaggatt tcatgctatt cttacttcca catctccagc ctccatctcc tggtaacagt 1620 ctctcttcct tccagaccct ctctgtttct gtctcagtat ttttctctct tctccagctc 1680 agcttaagaa tgtttcacca tttttatttc ctcctcccag atctccccat atctcacttc 1740 ccctccctcc atcctcttcc tccatctctc tctttctctc cccacttcct acccttcctc 1800 catggagtat ccccttatcc ctctgtttct ctgcgcatct ctatctggcc tttctgtttc 1860 tcttctgatt ctcttattct ttctacccgg tctctctctc tctctctctc tctctctctc 1920 tctctctctc tctctctctc tctctctctc tctccctcgt gtttctctga ctgagtttgc 1980 acctgtcctg ggcactggca ctgaatccat ctctctccca ccccactacc cctctgctcc 2040 acccttgggg agccccttgg aactggtccg ctcctgccac cgccgccccc tgacctctct 2100 ccacccccgc cttgacctcc ccaggcgtgg cgttcagcaa cggggagcgc gccaagcagc 2160 tcctgcgctt tgccatcgcc accctgaggg acttcggggt gggcaagcga ggcatcgagg 2220 agcgcatcca ggaggagtcg ggcttcctca tcgaggccat ccggagcacg cacggtgagt 2280 aaggttcccc gagtgcgggg gcaggagaag gaaaacaccc aggacgagga acccgcgcgc 2340 gttctgcctg cggatgggga ctaggtgggg aaaggcgccc gcacttccag ccctggagtc 2400 tggcgctggg aatttggctc aacaaggccc tgcctcctgg aattctgatt ctcctcagac 2460 ctctgagttg actctctccc caaccccctt ctccctccac acccgcaggc gccaatatcg 2520 atcccacctt cttcctgagc cgcacagtct ccaatgtcat cagctccatt gtctttgggg 2580 accgctttga ctatgaggac aaagagttcc tgtcactgct gagcatgatg ctaggaatct 2640 tccagttcac gtcaacctcc acggggcagg taactggctg cagcccggcc cgtgacgccc 2700 ctaccacaac ctgccaactg ctcccctacc tggagacagg tgccccaaac tcccaccccg 2760 ctccagacag tgtcccctca aaatcagccc ccgatattgg acaactggac agttgcacca 2820 gaagcctgtc tccaaggaca cctggatagc tcaacagatg ctccccaaaa cagagcctgc 2880 tggtaggata cataccctca gctcagctct ctcacctggg cacgtgttcc catccccaac 2940 ttaccgtaat ttctaacaga tgctccctac ccagttcttc tgaatatttt aacacctgga 3000 caaatgactg cgtcaacccg cctcctgcat gcctgaacac ctggtgctgc aaaatccagc 3060 ccatcataat catttcacat ctacacaaat gtcccagatt agcccactgg aatatctggc 3120 caagcgccct ctacttcacc cacttaaatg cctgaaaaca tggacagctg ccctaaccaa 3180 caccttaaac acacaaatat ctagacagat tgttccctga cacacaaata agtgttcccc 3240 aaccctttcc aatcacacac cttacagagg tgctccctgt gcatcgcact tggataggta 3300 aacacctcaa caggtatccc ctgcacttca acatcttcac cagccccact ttaatacctg 3360 aacacctgaa caaaagcccc caatccagac ccagtaagta tctggacagc tgtctccaac 3420 caagcctact tgaatgccta aatacctaga caggtgacac tcacctcata ccagccccac 3480 ctgaagagct aaacacctgg acaggtgtct tccaactcaa cttcacttga atatctgaac 3540 acctagatgt gtgctccaat ccagcctcat ttgcatacct gaaacctgga tatatgcctc 3600 agttcttctc acctaaatta ctagaccgtg gccctggtac ctaaccttcc tgaaaactta 3660 gatataagtt cctatccgac cccactgaaa tacctaaaca atgagacaga tgcctttaac 3720 tcagttcctt ccttgctatg aaacaaatcc cattcccatc agctcctgcc ccgtgacagc 3780 tgtccttccc ttcccatcct ctctctgcaa ccccagctct atgagatgtt ctcttcggtg 3840 atgaaacacc tgccaggacc acagcaacag gcctttaagt tgctgcaagg gctggaggac 3900 ttcatagcca agaaggtgga gcacaaccag cgcacgctgg atcccaattc cccacaggac 3960 ttcatcgact cctttctcat ccacatgcag gaggtacacc ccagcagcca gtgcggggag 4020 gtgcaaagcc aggcagaggg aaatcagact gggagtgggg cgggcagacg acacagaccc 4080 gttcaaatta gccctcatca taataatcct cacaattggc tgggcgccgt ggctaacagc 4140 ctgtaatccc agcactttgg gaggccgagg caggtggatc acctgaggtc aggagttcaa 4200 gaccagcctg gccaacatgg tcaaaccctg tctctactaa aaatccaaaa attagctgca 4260 cgtggtggcg cgaagggggg cagaggttgc aatgagccaa gatcacggca ctgcactcca 4320 gcctgggtga cagaatgaga ccctgtctca aaaaaaatta attaattgtt taaaaagtaa 4380 gtgagcctgc atggtcatgc gcgtgtgcag ttccagctac tcaggaggct aagggtggag 4440 gattgcttga gcccaggagt tcgagtccag cctgtgcaac ttagcaagac caagtcagtg 4500 taagaaaaaa aaaactgaca gctaagttga taattaaagg acagatggtc agcaacgtaa 4560 agaaggtgag aagaaagagc attttgggca aagccagcag ccagggcaag ggctggaacc 4620 tagagcgagt ctggtagatc tagggtccct ctttccacct ttggtctgga ccaaagagag 4680 gtagatccaa aggaaaagcc ctagaagggc cctgagggca agaggagtga ggttggccta 4740 aagccccctc tccctgcagg aggagaagaa ccccaacacg gagttctact tgaagaacct 4800 gatgatgagc acgttgaacc tcttcattgc aggcaccgag acggtcagca ccaccctgcg 4860 ctatggcttc ttgctgctca tgaagcaccc agaggtggag ggtaaggctg gagggggacg 4920 gaagtggagg gccccagacc ctcaaaattc cccttcgact ggtgcaatgt ccctacctgt 4980 cccagatccc aggaccctga gacgtgcctt gctgtccaga gacagggcaa cattcagctg 5040 gtaggcatca gctgagtctc attagctatt aaaatattga aaatgtctgc actgattggt 5100 cagtcacttc tgtcccaagc ccactgagtg cccgctgccc cttcccccgg gtcatctcct 5160 aagttcctcc ctgtgcctcc cctgtgattc tggcacaacc tggttaacag gatcctactc 5220 caacaatgcg aatggccgat gtctgttctg ttatgaatgc tctacctccg tctcataggg 5280 ggagccgtat catccacccc gttttaccta ttcagactat catttcctgc tctgagactc 5340 ccagatacct aaacacattc cccctcctcc cccagccaag gtccatgagg agattgacag 5400 agtgatcggc aagaaccggc agcccaagtt tgaggaccgg accaagatgc cctacatgga 5460 ggcagtgatc cacgagatcc aaagatttgg agacgtgatc cccatgagtt tggcccgcag 5520 ggttaaaaag gacaccaagt ttcgggattt tttcctccct aaggtgctat cctccccacc 5580 ccctagacta cggggacttc cagcccttct ctgtgtcccc agaatcctgc ccccattaga 5640 agctttctac tcactgtccc actccctcaa tcagtcaaaa aggacttccc caaccaccac 5700 atccattcca cctttccact tagacactcc tgagtcctgc atctccccag actctttgtg 5760 tcgggagaat caaactcaag ttccaaaact tcctatctta agaaacagaa gccccctttc 5820 cattaggcct tttgtcttag ggacacaaat ttcaggtccc ccaaacaacc tgcgtagcaa 5880 gacatggacc ccatgtctcc caaacttcct gtttcagaga tgtgaacctt ctattcccca 5940 aagctcctcc ttcacaggac cccaactcct ccatgcctgc cacttcccct tacctggggc 6000 acactagttc cccctccagc ccctgtgtac tttcaccaat cccccgaccc tcctcatcac 6060 acacaacttc ctcctcccta ccagggcacc gaagtgttcc ctatgctggg ctccgtgctg 6120 agagacccca gcttcttctc caaccctcag gacttcaatc cccagcattt cctggatgac 6180 aaggggcagt ttaagaagag tgatgctttt gtgccctttt ccatcggtaa gagaccactg 6240 tttgctgcca ggccactgct cacaccagca ggcgcctccc tcacccacct cccctctctg 6300 cggtgtagcc tggtatttct ccagcttgga agttcctgtt agaatctacc cttgagccag 6360 cagctgatac ttccttaact accaagcacc cagtacctgc acccaggtaa aaggaaagga 6420 aacatctttc cccgtagatg tatttctcta gggtcacaca gcagattcct cagatcccta 6480 aaaaggagat gacggcacag cagtcatatt tgcaagtgta tctggcaaga aggacatcta 6540 aacctcccat tgctacacct ggcatggatc accccatcta tgatggatgt gtgacattat 6600 gcctttttca aaacccatag aactgtataa cacagagtaa accctagtgt aaactatgga 6660 ctttgttagt aataatatat caatattggt tcaccattgt tacatctctt gtagaaagaa 6720 actgaggctc agggaggatc agagcctcct ctgaaactct ctcaggccat aatattccac 6780 ccctcctccc tgggagagcc gcagctggag gtcggtactg gggcgaggct gcactgagag 6840 tgggcttcac ctccacccct cccgcctctc ctcctcagga aagcggaact gtttcggaga 6900 aggcctggcc agaatggagc tctttctctt cttcaccacc gtcatgcaga acttccgcct 6960 caagtcctcc cagtcaccta aggacattga cgtgtccccc aaacacgtgg tctttgccac 7020 gatcccacga aactacacca tgagcttcct gccccgctga gcgagggctg tgccggtgca 7080 ggtctggtgg gcggggccag ggaaaggcgg ggtcagggcg gggttcgcgg aagaggcggg 7140 tataagaatg gggggaagat gcgggaaagg aaggggcgtg gtggctagag ggaagagaag 7200 aaacagaagc ggctcagttc accttgataa ggtgcttccg agctgggatg agaggaaggg 7260 aaaccttaca ttatgctatg aagagtagta ataatagcag ctcttatttc ctga 7314  

Claims (17)

1) extracting nucleic acids from biological samples taken from Koreans or Asians having similar genetic characteristics; 2) performing PCR with a primer capable of amplifying the CYP2A6 (cytochrome P450 2A6) gene or fragment thereof having the nucleic acid of step 1) as a template and having Korean or similar genetic characteristics; And 3) -48T> G in the nucleotide sequence of the PCR product obtained in step 2); 13G> A; 567C> T; 2134A> G; 3391T> C; 6458A> T; 6558T> C; 6582G> T; 6600G> T; And 6091C> T, 5971G> A and 5983T> G; Method for determining the genotype of the human CYP2A6 gene comprising the step of examining the presence of a CYP2A6 gene mutation consisting of. 1) extracting nucleic acids from biological samples taken from Koreans or Asians having similar genetic characteristics; 2) performing PCR with a primer capable of amplifying the CYP2A6 (cytochrome P450 2A6) gene or fragment thereof having the nucleic acid of step 1) as a template and having Korean or similar genetic characteristics; And 3) -48T> G in the nucleotide sequence of the PCR product obtained in step 2); 13G> A; 22C> T; 51G> A; 567C> T; 1620T> C; 1836G> T; 2134A> G; 3391T> C; 6458A> T; 6558T> C; 6582G> T; 6600G> T; And 6091C> T, 5971G> A, and 5983T> G. The method for determining the genotype of the human CYP2A6 gene comprising the step of examining the presence or absence of a single CYP2A6 gene mutation. 1) extracting nucleic acids from biological samples taken from Koreans or Asians having similar genetic characteristics; 2) performing PCR with a primer capable of amplifying the CYP2A6 (cytochrome P450 2A6) gene or fragment thereof having the nucleic acid of step 1) as a template and having Korean or similar genetic characteristics; And 3) -48T> G in the nucleotide sequence of the PCR product obtained in step 2); 22C> T; 51G> A; 567C> T; 1620T> C; 1836G> T; 3391T> C; 6458A> T; 6558T> C; 6600G> T; And 6091C> T, 5971G> A, and 5983T> G. The method for determining the genotype of the human CYP2A6 gene comprising the step of examining the presence or absence of a single CYP2A6 gene mutation. 1) extracting nucleic acids from biological samples taken from Koreans or Asians having similar genetic characteristics; 2) performing PCR with a primer capable of amplifying the CYP2A6 (cytochrome P450 2A6) gene or fragment thereof having the nucleic acid of step 1) as a template and having Korean or similar genetic characteristics; And 3) 22C> T in the nucleotide sequence of the PCR product obtained in step 2); 51G> A; 567C> T; 1620T> C; 1836G> T; 3391T> C; 6354T> C; 6458A> T; 6558T> C; 6600G> T; And 6091C> T, 5971G> A, and 5983T> G. The method for determining the genotype of the human CYP2A6 gene comprising the step of examining the presence or absence of a single CYP2A6 gene mutation. 1) extracting nucleic acids from biological samples taken from Koreans or Asians having similar genetic characteristics; 2) performing PCR with a primer capable of amplifying the CYP2A6 (cytochrome P450 2A6) gene or fragment thereof having the nucleic acid of step 1) as a template and having Korean or similar genetic characteristics; And 3) -48T> G in the nucleotide sequence of the PCR product obtained in step 2); 13G> A; 22C> T; 51G> A; 567C> T; 1620T> C; 1836G> T; 2134A> G; 3391T> C; 6458A> T; 6558T> C; And 6091C> T, 5971G> A, and 5983T> G. The method for determining the genotype of the human CYP2A6 gene comprising the step of examining the presence or absence of a single CYP2A6 gene mutation. 1) extracting nucleic acids from biological samples taken from Koreans or Asians having similar genetic characteristics; 2) performing PCR with a primer capable of amplifying the CYP2A6 (cytochrome P450 2A6) gene or fragment thereof having the nucleic acid of step 1) as a template and having Korean or similar genetic characteristics; And 3) -48T> G in the nucleotide sequence of the PCR product obtained in step 2); 13G> A; 22C> T; 51G> A; 567C> T; 1620T> C; 1836G> T; 3391T> C; 6458A> T; 6558T> C; 6600G> T; And 6091C> T, 5971G> A, and 5983T> G. The method for determining the genotype of the human CYP2A6 gene comprising the step of examining the presence or absence of a single CYP2A6 gene mutation. 1) extracting nucleic acids from biological samples taken from Koreans or Asians having similar genetic characteristics; 2) performing PCR with a primer capable of amplifying the CYP2A6 (cytochrome P450 2A6) gene or fragment thereof having the nucleic acid of step 1) as a template and having Korean or similar genetic characteristics; And 3) -48T> G in the nucleotide sequence of the PCR product obtained in step 2); 22C> T; 51G> A; 567C> T; 1620T> C; 1836G> T; 2134A> G; 3391T> C; 6458A> T; 6558T> C; 6600G> T; And 6091C> T, 5971G> A, and 5983T> G. The method for determining the genotype of the human CYP2A6 gene comprising the step of examining the presence or absence of a single CYP2A6 gene mutation. The method according to any one of claims 1 to 7, The biological sample of step 1) is selected from the group consisting of blood, skin cells, mucosal cells and hair. The method according to any one of claims 1 to 7, The primer of step 2) is characterized in that the primers of SEQ ID NO: 16, 17, 28 and 29. The method according to any one of claims 1 to 7, In the step 3), the presence or absence of the mutation is investigated using a snapshot analysis. 11. The method of claim 10, The snapshot analysis, characterized in that performed using a primer selected from the group consisting of the primers of SEQ ID NO: 18 to 27. delete delete delete delete delete delete

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