연구소 소식

학회명: 2009년도 한국분자•세포생물학회 정기학술대회 "RNA Biology and Disease"
주최기관: 한국분자•세포생물학회
기간: 2009.10.15-10.16
개최장소: 서울 코엑스 3층 컨퍼런스센터홀, 대서양홀8호
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[Symposium21] Molecular Systems Biology

[SY21-6] (Short Talk)
CYP2A6 Genotyping by a Multiplex Primer Extension Reaction

Yin-Jin Jang, In-Jun Cha, Jae-Gook Shin, Sang Seop Lee

Department of Pharmacology, Pharmacogenomics Research Center, Inje University College of Medicine, 633-165 Gaegum-dong, Jin-gu, Busan 614-735, Korea

Cytochrome P450 2A6 (CYP2A6) mediates the metabolism of nicotine as well as other xenobiotics and the activation of pro-carcinogens. Genetic studies have revealed that CYP2A6 gene, encoding CYP2A6 enzyme, is highly polymorphic regardless of ethnic populations. Previously, we studied the genetic polymorphism of CYP2A6 gene in Koreans and found that ten allelic variants of CYP2A6 in addition to wild-type are observed in Korean population. In the present study, we developed a powerful multiplex genotyping method for detection of all of these functional allelic variants of CYP2A6 using two rounds of PCR amplification and multiplex primer extension method. The alleles to be determined included CYP2A6*4, *7, *9A, *10, *11, *18A, *29,*30 and 567C>T (R101Stop). Except CYP2A6*4 that is a whole deletion of CYP2A6 gene, all of the other alleles were determined by SNaPshotTM analysis of an amplified 7.4kb-long CYP2A6 PCR product. For the detection of CYP2A6*4 allele, we amplified, sequenced and identified specific mutations linked to *4 allele. Using the linked mutations, the method of separate PCR amplification followed by single base primer extension was developed for genotyping *4 allele. Our CYP2A6 genotyping method consisted of two-rounds of PCR, one for *4 and one for the others, followed by two rounds of multiplex primer extensions reaction. The genotyping results of fifty Korean subjects were validated by the comparison with the results of direct sequencing, which showed 100% concordance. Compared to direct sequencing or agarose gel electrophoresis-based method, our method is more automatic, fast, economic, and reliable. Furthermore, considering the similarity of CYP2A6 polymorphism among Asians, our genotyping tools can be easily applied to other Asian populations.