Application of capillary gel electrophoresis to the diagnosis of the aldehyde dehydrogenase 2 genotype

ArticleinJournal of chromatography. B, Biomedical applications 685(1):185-90 · November 1996with4 Reads
DOI: 10.1016/0378-4347(96)00164-8 · Source: PubMed

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.

    • "...he laboratory setting (e.g., in the electrophoresis of DNA particles; Chiari et al., 1995; Tomita et al., 1996; Righetti and Gelf, 1997). No established exposure limits could be found in the literature for eithe..."
      More recent literature indicates the use of polymethacrylamide gels as biocompatible polymer matrices in gene and neuronal cell replacement therapy and for sustained-release drug formulations and drug targeting in the treatment of cancer (Pimm et al, 1996a,b; Chytry et al, 1996; Woerly et al., 1996a,b; Configliacchi et al., 1996; Wolfert et al, 1996; Liso et al., 1996; Plant et al., 1997). BAC is also currently used in the laboratory setting (e.g., in the electrophoresis of DNA particles; Chiari et al., 1995; Tomita et al., 1996; Righetti and Gelf, 1997). No established exposure limits could be found in the literature for either MAC or BAC.
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  • [Show abstract] [Hide abstract] 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.
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