APPLICATION OF CAPILLARY GEL-ELECTROPHORESIS TO THE DIAGNOSIS OF THE ALDEHYDE DEHYDROGENASE-2 GENOTYPE

This study dealt with the application of capillary gel electrophoresis (CGE) to diagnosis of the aldehyde dehydrogenase 2 (ALDH-2) genotype, Electrophoresis was performed on a low cross-linked polyacrylamide ge l {3% T [g acrylamide + g Bis (N,N'-methylenebisacrylamide)], 0.5% C ( g Bis/%T)} in 100 mM Tris-borate buffer (pH 8.3) at -10 kV with on-col umn UV detection (260 nm). During the PCR reaction, DNA from the wild- type allele generated a MboII restriction site, which is an amplificat ion created restriction site. This did not occur, however, with DNA fr agments from the mutant allele. Therefore, determination of the hetero zygous genotype, the coexistence of wild-type and mutant alleles, was easily possible. Analysis of the MboII restriction digests of the PCR products was completed in less than 20 min, showing two peaks correspo nding to fragments of 125 (cleaved) and 135 (uncleaved) base pairs (bp ), respectively. On the other hand, determination of the homozygous ge notype, wild-type or mutant, was difficult in one electrophoresis run. The CGE of the MboII restriction digests gave a single peak and the i dentification, cleaved or uncleaved, was difficult under our experimen tal conditions. However, the addition of aliquots of the PCR reaction mixture to the restriction digests, followed by re-electrophoresis, al lowed successful diagnosis, yielding two peaks (cleaved and uncleaved) for the wild-type and one peak (uncleaved) for the mutant allele. Thi s study demonstrated that CGE offers a high-speed, high-resolution ana lytical tool for determining genetic types, as compared with the conve ntional slab gel methodologies.