Real-time velocity of DNA bands during field-inversion gel electrophoresis.

The velocity v of bands of double-stranded, linear DNAs containing 48.5-5700 kbp was determined with 0.3 s resolution during field-inversion agarose gel electrophoresis (FIGE) for a broad range of the forward pulse period T+, keeping the duration of the backward pulse T- = T+/3. Within 0.6 s or less after the field changed sign from-to +, the velocity showed a sharp positive peak; a similar spike, but with negative velocity, occurred immediately after the field changed from + to -. For long pulses, the magnitude of this spike increased with M0.36, reaching ten times the steady-state velocity for M = 5.7 kbp. After this spike, the velocity dipped to 55-75% of its value in a steady field, then increased to a small secondary peak before reaching a steady-state plateau. The duration of the velocity trough, and the time of the small peak, increased as M1. For standard FIGE conditions (ratio of forward:reverse pulse duration, T+:T- = 3:1; equal forward and reverse field amplitudes, E+ = E-), the mobility mu = integral of vdt over a complete cycle was a minimum when E+ terminated at the end of the velocity trough. The minimum occurred because the velocity during E+ sampled primarily the trough, and because the backward velocity during E- was exceptionally large; the negative velocity spike was maximized when T+ terminated at the end of the velocity trough. Computer simulations of FIGE by Zimm (J. Chem. Phys. 1991, 94, 2187-2206) and by Duke and Viovy (J. Chem. Phys. 1992, 96, 8552-8563) generate real-time velocities that are in excellent agreement with our experimental data.