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Information on DNA conformation derived from the Ferguson plot of DNA fragments of up to 9 kb in size, using polyacrylamide gel electrophoresis in a discontinuous buffer system

Information on DNA conformation derived from the Ferguson plot of DNA fragments of up to 9 kb in size, using polyacrylamide gel electrophoresis in a discontinuous buffer system

Electrophoresis 12(4): 241-246

The Ferguson plot in polyacrylamide gel electrophoresis (PAGE) (15% CDATD, moving boundary electrophoresis buffer system operative at pH 8.9, 4.degree.C, 8 mA/cm2 of gel) of DNA fragments up to 9.4 kb in size was found to exhibit a linear segment at polyacrylamide concentrations starting at 3%T and undergoing a gradual transition into a concave segment at higher gel concentrations, confirming previous findings by Stellwagen [1]. The larger the DNA, and the higher the gel concentration, the less extended the linear and the more extended the concave segment of the plot. The lowest %T of the linear range for DNA in polyacrylamide remains unknown since mobilities at nongelling concentrations below 3%T have not as yet been measured. As previously suggested [1, 2], the transition from the linear to the concave segment corresponds to that from the randomly oriented DNA to the anisotropically stretched, "reptating" DNA. For a DNA of 9.4 kb in size, the end of the linear range of the Ferguson plot can be extended from 3.5 to 5%T when 15% DATD rather than 2.5% Bis is used to crosslink the polyacrylamide. Increasing the temperature of PAGE from 4.degree.C to 25 and 50.degree.C widens the linear segment progressively, indicating an increasingly random orientation with rising temperature. When current density is increased from 8 to 40 mA/cm2, the concave curvature f the Ferguson plot of DNA 1 to 9.4 kb in size decreases, suggesting a transition from a "reptating" to a randomly distributed molecule, due to increased Joule heat. Increasing the ionic strength from 0.01 to 0.04 M similarly appears to lead to a relative linearization of the Ferguson plot, suggesting a similar effect of Joule heat. Prestaining of DNA with ethidium bromide results in a decrease of both its size and its free mobility. This effect is exacerbated as the size of DNA increases from 0.1 to 9.4 kb.

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Accession: 040439256

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PMID: 1712721

DOI: 10.1002/elps.1150120403

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