Analytical Sciences

Abstract − Analytical Sciences, 30(11), 1093 (2014).

Simultaneous Genotyping of Single-nucleotide Polymorphisms in Alcoholism-related Genes Using Duplex and Triplex Allele-specific PCR with Two-step Thermal Cycles
Naoto SHIRASU and Masahide KUROKI
Department of Biochemistry, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan, Fukuoka 814-0180, Japan
We developed a time- and cost-effective multiplex allele-specific polymerase chain reaction (AS-PCR) method based on the two-step PCR thermal cycles for genotyping single-nucleotide polymorphisms in three alcoholism-related genes: alcohol dehydrogenase 1B, aldehyde dehydrogenase 2 and μ-opioid receptor. Applying MightyAmp® DNA polymerase with optimized AS-primers and PCR conditions enabled us to achieve effective and selective amplification of the target alleles from alkaline lysates of a human hair root, and simultaneously to determine the genotypes within less than 1.5 h using minimal lab equipment.