Detection and identification of cytochrome P-4502C9 alleles*1,*2, and*3 by high-resolution melting curve analysis of PCR amplicons

High-resolution melting curve analysis using a fluorescent DNA binding dye can detect sequence variations in a closed-tube system without labeled primers or probes. We developed and verified a melting analysis assay for common single nucleotide polymorphisms of cytochrome P-450 (CYP) 2C9 that affect warfarin metabolism. We used this method to genotype 84 patients receiving warfarin. For wild-type, *1/*1, 50% fluorescence corresponded to a mean +/- SD of 87.17 +/- 0.05 degrees C, whereas *2/*2 was 0.4 degrees C lower The *1/*2 melting curve was easily distinguished from *1/*1 and *2/*2 based on transition temperature and shape. Exon 7 showed a more complex melting curve; however, genotypes *1/*1, *1/*3, and *3/*3 were easily distinguishable. Melting curves were highly reproducible (SD of temperature for multiple fluorescence values 0.04 degrees C-0.11 degrees C; mean, 0.06 degrees C). Heterozygotes (*1/*2 or *1/*3) required significantly lower mean maintenance warfarin doses compared with wild-type (30.67 and 29.56 vs 42.81 mg/wk; P < .05). High-resolution melting analysis provides a simple and accurate method for genotyping of CYP2C9.