Makiko Shimizu1, Ren Sawaya1, Izumi Kishimoto1, Hiroshi Yamazaki2. 1. Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan. 2. Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan. Electronic address: hyamazak@ac.shoyaku.ac.jp.
Abstract
BACKGROUND: Genetic polymorphisms of human cytochrome P450 2A6 (CYP2A6) are one of the determinants of smoking behavior and/or tobacco-related lung cancer risk in male Japanese smokers. To help identify those at high risk, we developed a multiplex real-time polymerase chain reaction (PCR)-based genotyping method with dual-labeled probes to detect wild-type and whole-gene deletion of CYP2A6 directly from blood samples without DNA isolation. METHODS: We validated the new real-time PCR method that uses dual-labeled probes by utilizing 116 genomic DNA samples that had been genotyped previously and 33 blood samples. RESULTS: The new method could discriminate CYP2A6 from highly homologous CYP2A7 and CYP2A13 genes and could also determine CYP2A6*1 (wild type) and CYP2A6*4 (whole-gene deletion) alleles in perfect accordance with previous analysis data. Amplification curve profiles were obtained by multiplex real-time PCR assay with CYP2A6*1 and CYP2A6*4 primer sets and dual-labeled probes using one-drop blood samples previously genotyped for CYP2A6*1/*1, CYP2A6*1/*4, and CYP2A6*4/*4. CONCLUSIONS: A real-time multiplex PCR assay for genotyping wild-type CYP2A6 and whole-gene deletion was developed with dual-labeled probes. The new method achieved 100% agreement with data from the conventional PCR method for 116 genomic DNA samples and samples from 33 volunteers, thereby establishing its validity.
BACKGROUND: Genetic polymorphisms of humancytochrome P450 2A6 (CYP2A6) are one of the determinants of smoking behavior and/or tobacco-related lung cancer risk in male Japanese smokers. To help identify those at high risk, we developed a multiplex real-time polymerase chain reaction (PCR)-based genotyping method with dual-labeled probes to detect wild-type and whole-gene deletion of CYP2A6 directly from blood samples without DNA isolation. METHODS: We validated the new real-time PCR method that uses dual-labeled probes by utilizing 116 genomic DNA samples that had been genotyped previously and 33 blood samples. RESULTS: The new method could discriminate CYP2A6 from highly homologous CYP2A7 and CYP2A13 genes and could also determine CYP2A6*1 (wild type) and CYP2A6*4 (whole-gene deletion) alleles in perfect accordance with previous analysis data. Amplification curve profiles were obtained by multiplex real-time PCR assay with CYP2A6*1 and CYP2A6*4 primer sets and dual-labeled probes using one-drop blood samples previously genotyped for CYP2A6*1/*1, CYP2A6*1/*4, and CYP2A6*4/*4. CONCLUSIONS: A real-time multiplex PCR assay for genotyping wild-type CYP2A6 and whole-gene deletion was developed with dual-labeled probes. The new method achieved 100% agreement with data from the conventional PCR method for 116 genomic DNA samples and samples from 33 volunteers, thereby establishing its validity.