Detection of a point mutation in FAS2 gene of sake yeast strains by allele-specific PCR amplification

To identify yeast mutants with a point mutation, detection of the specific mutant alleles is necessary. For this purpose, we applied allele-specifie p olymerase chain reaction (PCR) to detect the FAS2-1250S dominant mutant all ele that encodes an altered fatty acid synthase in Japanese brewer's yeast strains. These strains are known to produce a higher amount of ethyl caproa te in Japanese sake. The mutant strains were supposed to be diploid and to contain heterozygous alleles, including wild-type FAS2 and a dominant FAS2- 1250S. A set of oligonucleotide primers was designed to contain different n ucleotides at their 3' termini: one type was identical to the wild type and the other to the mutant FAS2. Another set of primers was designed to have an additional mismatch at the second nucleotide from their 3' termini. By t esting with control strains, we established PCR conditions for specific amp lification. Using these conditions and a simple template preparation proced ure with SDS, the presence of the allele was detected in commercially used sake yeast strains. The method presented here will be useful for the identi fication of specific yeast strains.