High-resolution SSCP analysis using polyacrylamide agarose composite gel and a background-free silver staining method.
We describe here an improved methodology for single-strand conformation polymorphism (SSCP) analysis of PCR products. The method utilizes a polyacrylamide agarose composite gel and background-free silver staining. In comparison with conventional pure polyacrylamide gel for PCR-SSCP analysis, the composite gels have a much greater mechanical strength and improved resolution. The background staining commonly seen in many silver staining protocols has been eliminated by incorporation of thiosulfate, which can prevent nonspecific deposits of silver salts. As shown in our titration tests, the composite gel and background-free silver staining together have allowed clear identification of point mutations in samples containing as little as 5% of the target sequences, the same sensitivity achieved by radioactive labeling methods.
D004247:DNA / Q000032:analysis*; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D016158:Genes, p53; D006801:Humans; D008970:Molecular Weight; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D016622:Silver Staining / Q000379:methods*; D014407:Tumor Cells, Cultured
D004247:DNA