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 gel ¿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-column UV detection (260 nm). During the PCR reaction, DNA from the wild-type allele generated a MboII restriction site, which is an amplification created restriction site. This did not occur, however, with DNA fragments from the mutant allele. Therefore, determination of the heterozygous 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 corresponding to fragments of 125 (cleaved) and 135 (uncleaved) base pairs (bp), respectively. On the other hand, determination of the homozygous genotype, wild-type or mutant, was difficult in one electrophoresis run. The CGE of the MboII restriction digests gave a single peak and the identification, cleaved or uncleaved, was difficult under our experimental conditions. However, the addition of aliquots of the PCR reaction mixture to the restriction digests, followed by re-electrophoresis, allowed successful diagnosis, yielding two peaks (cleaved and uncleaved) for the wild-type and one peak (uncleaved) for the mutant allele. This study demonstrated that CGE offers a high-speed, high-resolution analytical tool for determining genetic types, as compared with the conventional slab gel methodologies.
Tomita
M
M
Department of Legal Medicine, Kawasaki Medical School, Kurashiki, Japan.
Okuyama
T
T
Hidaka
K
K
Ameno
S
S
Ameno
K
K
Ijiri
I
I
D000444:Aldehyde Dehydrogenase / Q000235:genetics*; D000071396:Aldehyde Dehydrogenase, Mitochondrial; D000483:Alleles; D004247:DNA / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific; D019075:Electrophoresis, Capillary; D004591:Electrophoresis, Polyacrylamide Gel; D005838:Genotype; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction
D004247:DNA; C096127:ALDH2 protein, human; D000444:Aldehyde Dehydrogenase; D000071396:Aldehyde Dehydrogenase, Mitochondrial; C024413:endodeoxyribonuclease MboII; D015252:Deoxyribonucleases, Type II Site-Specific