Conformation of Double-Stranded DNA during Agarose Gel Electrophoresis: Fractionation of Linear and Circular Molecules with Molecular Weights between 3 X 10⁶ and 26 X 10⁶

To answer several questions concerning the mechanisms of DNA fractionation during agarose gel electrophoresis, the electrophoretic mobility (μ) of double-stranded DNA has been measured as a function of (1) DNA topological conformation (linear, open circular, closed circular) and molecular weight (Mr) (molecular weights were between 2.9 X 10⁶ and 26.4 X 10⁶), (2) gel concentration (A) and temperature, and (3) voltage gradient. It was found that n extrapolated to an A of 0 (μ₀’) was independent of DNA conformation. The effect of temperature was to raise values of μ0’ in inverse proportion to buffer viscosity. Semilogarithmic μ vs. A plots for linear DNAs 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 DNAs, the negative slope (K_R) 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 μ vs. A plots were more linear for closed and open circular DNAs than they were for linear DNAs. Closed circular DNAs had K_R's lower than K_R's of either open circular or linear DNAs of the same molecular weight. At the lower voltage gradients, open circular DNA had the same K_R as linear DNA of the same molecular weight. However, as voltage gradient and molecular weight increased, the K_R of open circular DNA became smaller than the K_R of linear DNA (of the same molecular weight). This and the concave curvature of semilogarithmic μ vs. A plots for linear DNA resulted in a previously unreported reversal of the relative migration of linear and open circular DNAs as A increased.