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Distinction between supercoiled and linear DNA in transverse agarose pore gradient gel electrophoresis

Authors

  • Dr. David Wheeler,

    Corresponding author
    1. Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
    • Bldg.10, Rm. 6C101, NIH, Bethesda MD 20892, USA
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  • Jia-Hwei Lin,

    1. Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
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    • Department of Biochemistry, Eastern Virginia Medical School, Norfolk, VA.

  • Andreas Chrambach

    1. Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD
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Abstract

Four species of linear DNA and the first four members of a linking series, generated by treatment of plasmid DNA (PUC19, 2.7 kb) with mitochondrial topoisomerase, were differentiated by transverse agarose pore gradient gel electrophoresis. The experimental curves of migration distance vs. agarose concentration (Ferguson curves) of supercoiled DNA exhibit a steeper trajectory than those of linear DNA of the same size range. As a consequence, the four supercoiled species exhibit an increase in apparent size (relative to linear DNA standards) with increasing agarose concentration. Both the crossing of the Ferguson curves with those of linear standards as well as the apparent size increase with agarose concentration can serve to detect supercoiled plasmid-sized DNA in mixtures with linear DNA.

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