Metastable single-strand DNA conformational polymorphism analysis results in enhanced polymorphism detection.
Single-strand DNA conformational polymorphism (SSCP) makes use of sequence-dependent folding of single-stranded DNA (ssDNA), which alters the electrophoretic mobility of the fragments, to detect sequence differences between closely related molecules. In this study ssDNAs were purified by depletion of the complementary strand and PCR reactants on magnetic M-280-strepavidin beads. It was found that SSCP profiles created by purified ssDNAs differ from the profiles created by more usual SSCP methods. Under some conditions, SSCP profiles using whole PCR reaction products may result from the interaction between residual PCR primers and ssDNAs. We observed that the ratio of conformers revealed by band position and band intensity may vary between the assay techniques and misinterpretation of sequence variants may result. Another observation of this study was the formation of metastable conformational isomers with bead-purified ssDNAs by eliminating the thermal treatment used in conventional SSCP methods. The metastable SSCP (mSSCP) represents a novel and sensitive system for detection of sequence variation between closely related DNAs. The technique used here for the preparation of the purified ssDNAs is potentially useful for automated PCR-SSCP analysis using capillary electrophoresis or other methods.
Kasuga
T
T
Department of Molecular and Cell Biology, University of Aberdeen, UK.
Cheng
J
J
Mitchelson
K R
KR
D001345:Autoradiography; D001426:Bacterial Proteins; D001483:Base Sequence; D017931:DNA Primers; D004271:DNA, Fungal / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008280:Magnetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D011124:Polyporaceae / Q000235:genetics*; D012680:Sensitivity and Specificity; D019809:Streptavidin; D013462:Sulfur Radioisotopes
D001426:Bacterial Proteins; D017931:DNA Primers; D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D013462:Sulfur Radioisotopes; D019809:Streptavidin