Detection of a single base mismatch in double‐stranded DNA by electrophoresis on uncrosslinked polyacrylamide gel

Abstract

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.

Structure and mechanism for DNA lesion recognition

“…Indeed in the crystal structures of oligonucleotides containing a wobble base pair, base stacking in the neighborhood of the mismatch is perturbed and helical parameters are altered [39]. NMR studies find that a G/T mismatch causes local flexibility [42], and electrophoresis using uncrosslinked polyacrylamide gel revealed that DNA with a single mismatch migrates differently from normal DNA and resembles kinked species [43].…”

Generation of co‐dominant PCR‐based markers by duplex analysis on high resolution gels

“…The physical basis for the altered electrophoretic mobility is presumably the difference in the extent of DNA bending (Ganguly et al . 1993;Pulyaeva et al . 1994)…”

“…Finally, we have directly verified that different mobilities of various homoduplex fragments can arise from single nucleotide substitutions (Figures 1a and 2c,d). The physical basis for the altered electrophoretic mobility is presumably the difference in the extent of DNA bending (Ganguly et al, 1993;Pulyaeva et al, 1994) © Blackwell Science Ltd, The Plant Journal, (1998), 16, 117-125 Application of duplex analysis markers Analysis of CAPS markers. Gene mapping and positional cloning in Arabidopsis thaliana benefited tremendously from the development of CAPS markers (Konieczny and Ausubel, 1993).…”

C1 Inhibitor Gene Sequence Facilitates Frameshift Mutations

“…Precisely how the unpaired bases are involved is not yet clear. They may contribute simply by the helical distortion (30,31) or flexibility they induce, causing an unfavorable milieu for the DNA replication machinery. These sites may also be tagged by protein…”