Simultaneous genotyping of single-nucleotide polymorphisms in alcoholism-related genes using duplex and triplex allele-specific PCR with two-step thermal cycles.
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.
Department of Biochemistry, Faculty of Medicine, Fukuoka University.
Naoto
N
Shirasu
Masahide
M
Kuroki
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000437:Alcoholism / Q000235:genetics*; D000444:Aldehyde Dehydrogenase / Q000235:genetics; D000483:Alleles; D001483:Base Sequence; D003362:Cost-Benefit Analysis; D060005:Genotyping Techniques / Q000191:economics / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017450:Receptors, Opioid, mu / Q000235:genetics; D013696:Temperature; D013997:Time Factors
D017450:Receptors, Opioid, mu; D000426:Alcohol Dehydrogenase; D000444:Aldehyde Dehydrogenase