Application of the pyrosequencing technique for detection of VKORC1 and CYP2C9 genotypes / 国际检验医学杂志

ABSTRACT

Objective To establish genotyping methods for vitamin K epoxide reductase complex subunit 1 (VKORC1)and cytochrome P450 2C9(CYP2C9)based on pyrosequencing technique to detection of warfarin metabolizing enzyme related gene polymorphisms.Methods A total of 50 peripheral blood samples from healthy adults were collected and the whole blood genomic DNA was extracted.A set of biotin-labeled amplifi-cation primers and sequencing primers were designed respectively for three SNP sitesVKORC1 -1639 G>A,CYP2C9 430C> T and CYP2C9 1075A>C.After PCR amplification of the samples,pyrophosphoric acid se-quencing was conducted.And then the signal peaks form were combined to analyze and determine each sample genotype.Genotyping results were verified by Sanger sequencing,and the consistency of the two sequencing methods was compared.Results Genotypes of the three SNPs can be clearly determined according to the ba-ses and height of the signal peaks.Among the 50 samples,there were 41 AA and nine AG for VKORC1 -1639G>A,accounting for 82% and 12% respectively,and there were 45 *1/*1,five *1/*3 for CYP2C9, accounting for 90% and 10% respectively,no CYP2C9*2 allele detected.Genotype results detected by pyrose-quencing and Sanger sequencing were consistent with each other.Conclusion In SNP genotyping,Pyrose-quencing has the advantages of convenience,time-saving,cheap with accurate and reliable results,which can quickly determine the genotypes of CYP2C9 and VKORC1.