Temperature and pH effects on single-strand conformation polymorphism analysis by capillary electrophoresis.
We investigated the effects of temperature and pH on single strand-conformation polymorphism (SSCP) analyzed by capillary electrophoresis (CE) using short-chain linear polyacrylamide as the sieving medium. Nine different mutations (in factor V, cystathionine beta-synthase, and methylenetetrahydrofolate reductase genes), including both transitions and transversions, were investigated. We confirmed that low temperature in general increased the number of detectable single-strand conformations and thereby the sensitivity of the analysis. The pH effects of the separation matrix on the migration pattern, and thus the assay sensitivity, varied markedly between the different DNA fragments. Seven of nine single point mutations were detected at the ordinary pH of 8.3, whereas the CBS T833C mutation was discriminated at the extreme pH values of 9.0 and 6.4, and the CBS G797A mutation could not be detected at any pH value within the range 6.4--9.0. These data emphasize the importance of the pH of the separation matrix in detecting certain mutations by SSCP.
Ren
J
J
Department of Pharmacology, University of Bergen, Norway. jicun.ren@farm.uib.no
Ueland
P M
PM
D001483:Base Sequence; D002021:Buffers; D004252:DNA Mutational Analysis / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D013997:Time Factors
D002021:Buffers