Real-time genotyping of cytochrome P4501A1 A4889G and T6235C polymorphisms

Single nucleotide polymorphisms, such as the cytochrome P450 1A1 (CYP1A1) A4889G and T6235C variants, have been reported to be associated with an increased cancer risk. In order to study their role in molecular-epidemiological studies, we developed a single-step procedure for genotyping these two CYP1A1 polymorphisms using real-time polymerase chain reaction (PCR) and subsequent melting curve analysis. Genotypes of 300 unrelated Caucasians, without prior history of cancer, were determined by real-time PCR and compared to genotypes obtained by restriction fragment length polymorphism PCR. In the population examined, the allele frequency of the CYP1A1 G allele at position 4889 was 0.042, and the frequency of the T allele at position 6235 was 0.10. Five percent of samples disagreed between methods. Sequence analysis of discordant samples revealed that the differences are likely attributed to incomplete digestion of amplicons by the RFLP method. These findings confirm the reliability and utility of real-time PCR as a method for large-scale genotyping.