Conformation of double-stranded DNA during agarose gel electrophoresis: fractionation of linear and circular molecules with molecular weights between 3 .times. 106 and 26 .times. 106

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Abstract
To answer several questions concerning the mechanisms of DNA fractionation during agarose gel electrophoresis, the electrophoretic mobility (.mu.) of double-stranded [plasmid pBR322, yeast plasmid pYE(CEN3)41, bacteriophages T7 and .lambda. deletion mutant 590] DNA was measured as a function of DNA topological conformation (linear, open circular, closed circular) and MW (MW were between 2.9 .times. 106 and 26.4 .times. 106), gel concentration (A) and temperature and voltage gradient. .mu. extrapolated to an A of 0 (.mu.0'') was independent of DNA conformation. The effect of temperature was to raise values of .mu.0'' in inverse proportion to buffer viscosity. Semilogarithmic .mu. vs. A plots for linear DNA had curvature that was opposite to the curvature for spherical particles (plots for linear DNA were concave). As A approached 0, the plots became increasingly linear. For the larger DNA, the negative slope (KR) in the region of linearity was decreased as voltage gradient increased. These and other data indicate deformation of linear DNA random coils during agarose gel electrophoresis. The data suggest both an asymmetric and a symmetric collapse of linear DNA random coils during agarose gel electrophoresis. However, end-first migration of linear DNA, previously suggested by others, does not explain the data. The semilogarithmic .mu. vs. A plots were more linear for closed and open circular DNA than they were for linear DNA. Closed circular DNA had KR lower than KR of either open circular or linear DNA of the same MW. At the lower voltage gradients, open circular DNA had the same KR as linear DNA of the same MW. However, as voltage gradient and MW increased, the KR of open circular DNA became smaller than the KR of linear DNA (of the same MW). This and the concave curvature of semilogarithmic .mu. vs. A plots for linear DNA resulted in a previously unreported reversal of the relative migration of linear and open circular DNA as A increased.