SYBR Green-based real-time PCR assay for detection of VKORC1 and CYP2C9 polymorphisms that modulate warfarin dose requirement
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Sheng-Wen Huang
Abstract
Background: Polymorphisms in VKORC1 (vitamin K epoxide reductase complex subunit 1) and CYP2C9 (cytochrome P450 2C9) genes are considered the major genetic factors modulating warfarin dose requirement. As a result, a rapid and economic method for genotyping patients for predicting warfarin dose could be useful in clinical applications.
Methods: In this study, we developed and validated a melting temperature (Tm)-shift genotyping assay for detection of VKORC1 C1173T and CYP2C9 A1075C polymorphisms by using SYBR Green-based multiplex allele-specific PCR and consequent melting curve analysis. In this Tm-shift genotyping assay, a GC-tail was incorporated into one of the two allele-specific primers to increase the melting temperature of PCR product, such that both alleles can be differentially detected simultaneously at their respective melting curve patterns. In total, 100 randomly selected samples of both VKORC1 and CYP2C9 genes from each genotype group determined by melting curve were validated by direct DNA sequencing and denaturing high-performance liquid chromatography (DHPLC) analysis.
Results: There was 100% concordance between the results of DNA sequencing and Tm-shift genotyping, and 99% concordance between the results of DHPLC assay and Tm-shift genotyping.
Conclusions: This allele-specific PCR assay is convenient, inexpensive and will be useful for VKORC1 and CYP2C9 genotyping in a clinic laboratory.
Clin Chem Lab Med 2009;47:26–31.
©2009 by Walter de Gruyter Berlin New York
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