title: Genotyping of wild-type cytochrome P450 2A6 and whole-gene deletion using human blood samples and a multiplex real-time polymerase chain reaction method with dual-labeled probes.
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.
authors.affiliation: Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.
authors.forename: Makiko
authors.initials: M
authors.lastname: Shimizu
authors.affiliation: Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.
authors.forename: Ren
authors.initials: R
authors.lastname: Sawaya
authors.affiliation: Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan.
authors.forename: Izumi
authors.initials: I
authors.lastname: Kishimoto
authors.affiliation: Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan. Electronic address: hyamazak@ac.shoyaku.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yamazaki
mesh_terms: D000328:Adult; D001800:Blood Specimen Collection; D065637:Cytochrome P-450 CYP2A6 / Q000097:blood* / Q000235:genetics*; D005260:Female; D017353:Gene Deletion; D005838:Genotype; D006801:Humans; D008297:Male; D008875:Middle Aged; D015341:Nucleic Acid Probes / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction; D055815:Young Adult
chemical_list: D015341:Nucleic Acid Probes; D065637:Cytochrome P-450 CYP2A6
keywords: CYP2A6; Genotyping; Multiplex PCR; Whole-gene deletion; Wild type