Duplex genotyping of CYP2C19*2 and CYP2C19*3 by high-resolution melting curve analysis

Abstract

Clopidogrel is a widely used anti-platelet agent for the prevention of arterial thrombosis. It has been suggested that clopidogrel may be less effective in inhibiting platelet aggregation among patients who are carriers of CYP2C19*2 and CYP2C19*3, two loss-of-function CYP2C19 alleles, which are associated with reduced conversion of clopidogrel to its active metabolite. The objective of this research was to develop a simple and accurate method for genotyping of CYP2C19*2 and CYP2C19*3 simultaneously in one closed-tube using high-resolution melting curve (HRM) analysis. Two amplicons bracketing CYP2C19*2 and CYP2C19*3 gene variants were designed, and AT- or GC-rich 5' tails were added to selected primers to ensure two different amplicons with non-overlapping melting curves. Sixty-four random DNA samples were all fast and sensitively genotyped by HRM analysis. This method was validated by DNA sequencingtechnique, and genotypes obtained using the HRM approach perfectly matched the genotypes obtained by DNA sequencing technique. Therefore, this HRM-based assay allows simple and accurate duplex genotyping of CYP2C19*2 and CYP2C19*3 simultaneously in one closed-tube. This method is expected to be applied in clinical laboratory to guide indi-vidual dosage design of clopidogrel.