METASTABLE SINGLE-STRAND DNA CONFORMATIONAL POLYMORPHISM ANALYSIS RESULTS IN ENHANCED POLYMORPHISM DETECTION

Single-strand DNA conformational polymorphism (SSCP) makes use of sequ ence-dependent folding of single-stranded DNA (ssDNA), which alters th e electrophoretic mobility of the fragments, to detect sequence differ ences between closely related molecules. in this study ssDNAs were pur ified by depletion of the complementary strand and FCR reactants on ma gnetic M-280-strepavidin beads. It was found that SSCP profiles create d by purified ssDNAs differ from the profiles created by more usual SS CP methods. Under some conditions, SSCP profiles using whole PCR react ion products may result from the interaction between residual PCR prim ers and ssDNAs. We observed that the ratio of conformers revealed by b and position and band intensity may vary between the assay techniques- and misinterpretation of sequence variants may result. Another observa tion of this study was the formation of metastable conformational isom ers with bead-purified ssDNAs by eliminating the thermal treatment use d in conventional SSCP methods. The metastable SSCP (mSSCP) represents a novel and sensitive system for detection of sequence variation betw een closely related DNAs. The technique used here for the preparation of the purified ssDNAs is potentially useful for automated PCR-SSCP an alysis using capillary electrophoresis or other methods.