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

• A multiplex real-time polymerase chain reaction (PCR) was developed for CYP2A6.
• Wild-type and whole-gene deletion of CYP2A6 were determined from blood samples.
• Two primer sets and two dual-labeled probes generated amplification curve profiles.
• Reliability of the method was validated using the conventional PCR method.
• This simple method will support individual counseling for cigarette cessation.

BackgroundGenetic 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.MethodsWe 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.ResultsThe 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.ConclusionsA 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.

Multiplex real-time polymerase chain reaction method with dual-labeled probes for genotyping of CYP2A6.Figure optionsDownload as PowerPoint slide