Direct genotyping of Cytochrome P450 2A6 whole gene deletion from human blood samples by the SmartAmp method.

ABSTRACT

BACKGROUND: Genetic polymorphisms of the human CYP2A6 gene are considered to be a determinant of smoking behavior and tobacco-related lung cancer risk in male Japanese smokers. We developed a SmartAmp-based genotyping method to detect whole deletion of the CYP2A6 gene directly from blood samples without DNA isolation. METHODS: We validated the new method using CYP2A plasmids, 48 genomic DNA samples and 25 blood samples by utilizing the SmartAmp method, a unique isothermal DNA amplification process. RESULTS: This method could discriminate the CYP2A6 gene from highly homologous CYP2A7 and CYP2A13 genes. CYP2A6*1 (wild-type) and CYP2A6*4 (whole gene deletion) were determined by the new method in perfect accordance with sequence analysis data. CONCLUSIONS: A SmartAmp assay for genotyping the CYP2A6 gene was developed, and the reliability of the method was validated using the conventional PCR method.