Analytical Sciences

Abstract − Analytical Sciences, 32(12), 1363 (2016).

Duplex PCR-RFLP for the Simultaneous Genotyping of Single Nucleotide Polymorphisms in ADH1B and ALDH2 Genes
Naoto SHIRASU and Shin’ichiro YASUNAGA
Department of Biochemistry, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan, Fukuoka 814-0180, Japan
Single nucleotide polymorphisms (SNPs) in ADH1B and ALDH2, which encode alcohol dehydrogenase 1B and aldehyde dehydrogenase 2, respectively, are responsible for ethanol-metabolizing activity and alcohol-related disease. We developed a simple and rapid polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) method for the simultaneous genotyping of the genes in a single-tube reaction. Using an alkaline lysate of a human hair root as a template, SNP-containing regions of each gene were PCR-amplified simultaneously. The PCR products were directly applied to double restriction digestion, followed by agarose gel band analysis. The duplex PCR-RFLP allows for the simultaneous and accurate determination of the SNP genotypes of ADH1B and ALDH2 within 1.2 h.