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
Orbán, L. and Chrambach, A. (1991) 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). pp. 241-246.
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The Ferguson plot in polyacrylamide gel electrophoresis (PAGE) (15 %CDATD, moving boundary electrophoresis buffer system operative at pH 8.9, 4°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 . 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°C to 25 and 50°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 of 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.
|Publication Type:||Journal Article|
|Publisher:||John Wiley & Sons Ltd|
|Copyright:||© 1991 VCH Verlagsgesellschaft mbH|
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