Two‐dimensional motion of DNA bands during pulsed‐field gel electrophoresis. II. Effect of field angle

Abstract

AbstractIn pulsed‐field gel electrophoresis, megabase DNAs are well separated if the angle θ between the two electric fields is 120° but not if θ = 90°. To elucidate the molecular basis for this observation, we measured the instantaneous position (x, y) and velocity (vx, vy) of a band of G‐DNA (670 kb) while the field switched direction, for 90° ≤ θ ≤ 102°. For θ = 120° and long pulse period T. The band retraced the last segment of the preceding pulse before moving in‐ the new field direction. The retracing wax done at a velocity much greater than the average forward velocity. For θ = 90°, rather than retrace itself, the path during one pulse appeared to originate from a point beyond that reached in the previous pulse, end the velocity showed only a brief backward spike.A Monte Carlo simulation that included tube‐length fluctuations and hernias was carried out for a model DNA chain moving through a three‐dimensional network of interconnected pores, with parameters corresponding to the DNA size, agarose concentration, and field strength of the experiments, Both the xy path and the instantaneous velocities of the simulation were in excellent agreement with experiment for 90° ≤ θ ≤ 120°. When the field changed direction in the simulation, hernias often advanced from both ends in the new field direction. In the 120° case, those near the erstwhile trailing segments of the chain soon established superiority because chain tension and a component of the new field aided their growth. For θ = 90° and long T, however, segments from the head end were more likely to continue to lead because there was often an excess of relaxed segments there, and no component of the field aided either end. © 1995 John Wiley & Sons, Inc.