A wax-mediated hot-start polymerase chain reaction improves the genotyping of CYP2C9* 1 and * 3 alleles

Cytochrome P4502C9 (CYP2C9) is a polymorphic enzyme which metabolizes a variety of medications. The CYP2C gene families are extensively homologous; in particular, the nucleotide sequences in the areas adjacent to polymorphic sites are > 95% identical. Frequently, primers designed for amplification of CYP2C9 also amplify other CYP2C genes. Additional amplification of the other CYP2C alleles will thus yield an erroneous restriction pattern, resulting in misleading genotype of CYP2C9. Thus, a procedure to specifically amplify CYP2C9 alleles is necessary. In this study, a wax-mediated hot-start polymerase chain reaction (PCR) for the amplification of CYP2C9* 1 and * 3 alleles was tested. When serially diluted template DNA was amplified by the hot-start PCR followed by the agarose electrophoresis without the enzyme digestion, the 165 bp band derived from CYP2C9 sequence was clearly detected without any non-specifically amplified band even from 10 ng DNA template, and the density of this band increased in proportion to the amount of template. On the other hand, when the PCR without the use of wax was performed, the amplification of the allele was irregular. This hot-start PCR gave a significant increase in the sensitivity for the detection of CYP2C9* 1 and * 3 alleles. The performance of the present method was studied with 64 Japanese volunteers. After the enzyme digestion of the products obtained by the wax-mediated hot-start PCR, only specific bands corresponding to CYP2C9* 1 and * 3 alleles were obtained. This PCR method gave the consistent performance and low background. Thus, the wax-mediated hot-start PCR method should be useful in the genotyping of CYP2C9* 1 and * 3, possibly including CYP2C9* 2 allele in the clinical laboratory.