The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions.
Single-strand conformation polymorphism (SSCP) analysis has proven to be a simple and effective technique for the detection of single base substitutions. We have used SSCP to analyze 29 mouse globin mutations, 27 p53 mutations, and 8 rhodopsin mutations contained within different size PCR products. Our results indicate that the type of mutation (transition versus transversion) did not play a major role in determining whether a mutation was detected by SSCP analysis. The position of the base substitution was more important than the precise base substitution in determining whether a mutation was detected. We report that SSCP sensitivity varies dramatically with the size of the DNA fragment being analyzed. The optimal size fragment for sensitive base substitution detection by SSCP is approximately 150 bp. Our results illustrate the need to keep the size of the PCR fragment small when performing SSCP to detect mutations. Larger fragments can be analyzed when screening for polymorphisms when the need to detect every sequence variation is not as critical.
Sheffield
V C
VC
Department of Pediatrics, University of Iowa, Iowa City.
Beck
J S
JS
Kwitek
A E
AE
Sandstrom
D W
DW
Stone
E M
EM
D000818:Animals; D004252:DNA Mutational Analysis / Q000379:methods*; D004277:DNA, Single-Stranded / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D016158:Genes, p53; D005914:Globins / Q000235:genetics; D006801:Humans; D051379:Mice; D009154:Mutation; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011401:Promoter Regions, Genetic; D012243:Rhodopsin / Q000235:genetics; D012680:Sensitivity and Specificity
D004277:DNA, Single-Stranded; D005914:Globins; D012243:Rhodopsin