Molecular sieving of lambda phage DNA in polyacrylamide solutions as a function of the molecular weight of the polymer

Abstract

Electrophoresis of lambda phage DNA was carried out in solutions at various concentrations of uncrosslinked polyacrylamide of 0.6, 1, 5 and 9 × 10⁶ molecular weight (M_w) with narrow M_w distribution. By inspection of mobilities in the various concentration ranges, it appears that mobilities decrease, and retardation increases, with increasing M_w. The relation between electrophoretic retardation and the M_w of the polymer was also interpreted (i) in the manner previously applied to nonlinear Ferguson plots and compatible with the Ogston model; and (ii) empirically, on the basis of the first derivatives of the functions describing the Ferguson plots at the polymer concentrations used. Interpretation (i) shows that the retardation increases linearly in the order of 0.6, 1, 5 and 9 × 10⁶M_w of polyacrylamide. Interpretation (ii) shows a nonlinear increase of retardation in the M_w range 5 to 9 × 10⁶, and a decrease in retardation as M_w is raised from 0.6 to 5.0 × 10⁶. Hypothetically, interpretation (ii) can be explained mechanistically by a progressive change, as the polymer size is increased, from a collision with the surface of the polymer fiber to one occurring after permeation in the interior of a randomcoiled fiber. Interpretation (i) may fail to detect that change due to the large difference between DNA mobility in solutions of the smallest polymer and the free mobility. DNA peak detection in all of the four size classes of polyacrylamide in solution is limited to relatively narrow ranges of polymer concentration. For the electrophoresis of lambda DNA, polyacrylamide of 5 × 10⁻⁶M_w represents an optimum due to a compromise between small mobility differences at various concentrations of the polymer with M_w 0.6 × 10⁶, either little retardation (interpretation (i)) or insufficient responsiveness of the Ferguson plot to concentration changes (interpretation (ii)) in presence of the polymer of 1 × 10⁶M_w and an excessively narrow available concentration range at 9 × 10⁶M_w. A highly disperse solution of polyacrylamide of 18 × 10⁶ molecular weight retarded lambda DNA only slightly. Retardation decreased upon ultrafiltration of the polymer solutions, presumably through the decrease of their concentrations or adsorption of the largest chains within the size distribution.