A study on ADH2 and ALDH2 genotyping by PCR-RFLP and SSCP analyses with description of allele and genotype frequencies in Japanese, Finn and Lapp populations.

Abstract

Genetic polymorphisms of the alcohol dehydrogenase ADH2 and aldehyde dehydrogenase ALDH2 genes were investigated in Japanese, Finn, and Lapp populations by using PCR-RFLP and SSCP analyses. The ALDH2 genotypes were unequivocally determined by a PCR-RFLP assay with a mismatched primer. The determination of the ADH2 genotypes, however, was found to be problematic in PCR with the reported oligonucleotide primer sets because there are high homologies among the ADHl, ADH2, and ADH3 gene sequences. The problem of the heterozygote excess in typing results obtained by using the previously reported PCR-RFLP methods was resolved by nested PCR, in which an internal primer set reamplified the ADH2 sequence selectively from a mixture of the ADH gene sequences amplified in the first PCR amplification of genomic DNA samples as templates. A newly designed primer pair with longer sequences and single 3' end mismatches was later found to achieve a predominant amplification of the ADH2 sequence in a single PCR. RFLP and SSCP analyses of PCR products with the new primer set gave results fully consistent with those by nested PCR. Thus, the ADH2 genotypes defined in this study were free from any typing errors. The ADH2 and ALDH2 allele frequencies observed in this study were found not to be biased significantly from those reported previously from Japanese populations, and these were monomorphic for Lapp and Finn populations.