Detection of a single base mismatch in double-stranded DNA by electrophoresis on uncrosslinked polyacrylamide gel.
Uncrosslinked polyacrylamide forms gels in the concentration range of 15-40% acrylamide. Electrophoresis in these gels of a commercially available 350 bp heteroduplex DNA preparation separates it from the homoduplex DNA of the same size. The separation is qualitatively equivalent to that previously achieved in a commercial proprietary gel ("Mutation Detection Gel" of AT-Biochem), or in an equivalent 14% T, 0.15% C (N,N'-methylenebisacrylamide) gel, but the mechanical stability of mutation detection electrophoresis (MDE) gels or 0.15% C gels is better than that of uncrosslinked polyacrylamide gels. The separation in any of these three gel media can be carried out in short gel tubes within a few hours of electrophoresis time. In both uncrosslinked polyacrylamide and MDE gel media, the Ferguson plots [log(mobility) vs. gel concentration] and the plots of effective molecular radius vs. gel concentration ("T-plots") of both the heteroduplex and homoduplex DNA indicate an augmented size but similar flexibility upon passage through the gel than exhibited by the components of a DNA standard ladder. Homoduplex and heteroduplex DNA correspondingly exhibit a parallelism of their Ferguson curves in transverse MDE pore gradient gel electrophoresis, suggesting a surface net charge difference, possibly due to a conformational reorientation too subtle to be detected by a shift in the slope of the Ferguson plot, as has been observed once previously with a "kinked" DNA species. The gel fiber radius or length per unit volume of uncrosslinked polyacrylamide and MDE gels do not differ significantly within confidence limits, which are wide compared to unconventionally crosslinked gels, presumably because of their greater swelling.
Division of Intramural Research, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD.
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H
Pulyaeva
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Zakharov
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MM
Garner
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A
Chrambach
D000180:Acrylic Resins; D001482:Base Composition; D003432:Cross-Linking Reagents; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D005782:Gels; D005821:Genetic Techniques; D007202:Indicators and Reagents; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry* / Q000302:isolation & purification; D000099166:Polyacrylamides
D000180:Acrylic Resins; D003432:Cross-Linking Reagents; D004247:DNA; D005782:Gels; D007202:Indicators and Reagents; D009692:Nucleic Acid Heteroduplexes; D000099166:Polyacrylamides