Application of capillary gel electrophoresis to the diagnosis of the aldehyde dehydrogenase 2 genotype.
ABSTRACT 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.
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ABSTRACT: Mutation detection plays a great role in genetic and medical research and clinical diagnosis of inherited diseases and particular cancers. Single-strand conformation polymorphism (SSCP) analysis is one of the most popular methods for detection of mutations. Recently, automated capillary electrophoresis (CE) systems have been used in SSCP analysis instead of conventional slab gel electrophoresis. SSCP analysis in combination with CE is a rapid, simple, sensitive and high-throughput mutation screening tool, and has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes. The new technique has a great potential for mutation screening of large numbers of samples in clinical diagnosis. This review discusses basic issues about the methodology of SSCP analysis based on CE and summarizes several key applications.Journal of chromatography. B, Biomedical sciences and applications 06/2000; 741(2-741):115-128. DOI:10.1016/S0378-4347(00)00090-6
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ABSTRACT: Timed-pregnant CD-1 outbred albino Swiss mice received either methacrylamide (MAC; 0, 60, 120, or 180 mg/kg/day) or N,N'-methylenebisacrylamide (BAC; 0, 3, 10, or 30 mg/kg/day) p.o. in distilled water on gestational days (GD) 6 through 17. Maternal clinical status was monitored daily. At termination (GD 17), confirmed-pregnant females (27-30 per group, MAC; 24-25 per group, BAC) were evaluated for clinical status and gestational outcome; live fetuses were examined for external, visceral, and skeletal malformations. For MAC, no treatment-related maternal mortality was observed. Maternal body weight on GD 17, maternal weight gain during treatment and gestation, and corrected maternal weight gain were reduced at the high dose. Relative maternal food and water intake was not adversely affected; neurotoxicity was not observed. Relative maternal liver weight was increased at > or = 120 mg/kg/day; gravid uterine weight was decreased at 180 mg/kg/day. The maternal no-observed adverse effect level (NOAEL) was 60 mg/kg/day. The NOAEL for developmental toxicity was also 60 mg/kg/day. At > or = 120 mg/kg/day, mean fetal body weight was reduced. At 180 mg/kg/day, increased postimplantation death per litter was observed. Morphological development was not affected. The maternal NOAEL for BAC was 10 mg/kg/day. At 30 mg/kg/day, decreased maternal body weight on GD 17, maternal body weight change during treatment and gestation, corrected maternal body weight, and gravid uterine weight were observed. Relative maternal liver weight increased at 30 mg/kg/day. The developmental NOAEL was 3 mg/kg/day BAC. Mean fetal body weight was reduced at 30 mg/kg/day. At > or = 10 mg/kg/day, an increased incidence of fetal variations (extra rib) was observed, although fetal malformation rate was unaffected. MAC and BAC were not teratogenic to Swiss mice at the doses tested. BAC was more potent than MAC in causing adverse maternal and developmental effects.Toxicological Sciences 12/1998; 46(1):124-33. DOI:10.1006/toxs.1998.2506 · 4.48 Impact Factor