Allele-Specific PCR Development for CYP2C9 Genotyping การพัฒนาเครื่องหมาย allele-specific PCR เพื่อระบุจีโนไทป์ของยีน CYP2C9

Abstract

CYP2C9 is an enzyme that has effects on pharmacokinetics of warfarin, an anticoagulant drug. This drug is a narrow therapeutic index drug because genetic polymorphisms of CYP2C9 have high effect on warfarin response. Genetic diversity of CYP2C9 with effects on therapeutic drug of warfarin were CYP2C9*2 (rs1799853) and CYP2C9*3 (rs1057910). There are several methods available for genotyping such as PCR-RFLP and real-time PCR. However, both methods are expensive, manifold steps and time consuming. Hence, the researchers aimed to develop an allele-specific polymerase chain reaction (AS-PCR) method for CYP2C9 genotyping. The expected PCR products of CYP2C9*2 were 154 bp (wild type allele), 230 bp (mutant allele) and 346 bp (control) whereas the expected PCR products of CYP2C9*3 were 128 bp (wild type allele), 200 bp (mutant allele) and 289 bp (control). The PCR reaction was optimized by adjusting annealing temperatures and MgCl₂ concentration. The results revealed that annealing temperature at 65^๐C and MgCl₂ 1.25 mM were the suitable PCR condition for both CYP2C9*2 and CYP2C9*3 genotyping. Sanger DNA sequencing method were used for result validation. In conclusion, AS-PCR developed in this study is a fast, simple and cost-effective technique to identify CYP2C9 genotype for warfarin dosage adjustment which is appropriate for the treatment.