Determination of human alcohol dehydrogenase and acetaldehyde dehydrogenase genotypes by single strand conformation polymorphism in discontinuous buffer electrophoresis.

Abstract

Under appropriate conditions single strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products allows the detection of single base mutations in a given DNA fragment. We adapted this method for the routine determination of allele variants of human alcohol and acetaldehyde dehydrogenase without radioisotopic labeling. After PCR amplification of the selected exon, the DNA fragments were heat-denatured and loaded on a polyacrylamide gel containing glycerol. For electrophoresis a discontinuous buffer system was used with sulfate as leading ion and borate as trailing ion. The DNA bands were revealed by silver staining. Acrylamide concentrations, ionic strength and electrophoresis temperature were systematically investigated for each DNA fragment. The polymorphisms detected by SSCP were identical to those found by hybridization with 32P-labeled allele-specific oligonucleotides. This method avoids the use of radioactivity, is less expensive and simpler than the allele-specific oligonucleotide (ASO) methodology and thus particularly suited for routine analysis.