دانلود High resolution melting method to detect single nucleotide polymorphism of VKORC1 and CYP2C9

Abstract

Single  nucleotide  polymorphisms  (SNPs)  of  VKORC  (1173T/C,  rs9934438)  and  CYP2C9  (1075A/C,  rs1057910) are major contributory factors on the sensitivity of warfarin in Chinese. Analysis of the two genomic loci could help warfarin treatment individual from bleeding or thrombosis events. An assay with the advantages of simplicity, speed, high sensitivity and low cost for genotyping is calling for clinical laboratories. High resolution method (HRM)  meets  these  callings,  but  no  study  with  large  sample  tested  its  performance  in  genotyping  of  rs9934438 and rs1057910. In this study, we identified polymorphisms of rs9934438 and rs1057910 in 255 unrelated Chinese heart valve replacement patients of Han ethnic group from West China Hospital. The two genomic loci were genotyped  by  HRM  using  LightCycler®  480  High  Resolution  Melting  Master  on  LightCycler®  480  Real-Time  PCR  instruments (Roche Diagnostics), and all amplified PCR products were sent for direct DNA sequencing. The genotyping of rs1057910 between HRM and sequencing showed perfect 100% concordance. While the concordance of rs9934438 between HRM and sequencing was 99.2%. Unexpected mutation interfered genotyping results of HRM when genotyping rs9934438. HRM is a valuable technique for genotype detection of rs9934438 and rs1057910 to assess individual sensitivity to warfarin, where DNA sequencing is added inevitably sometimes.