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 0.3s resolution during field-inversion agarose gel electrophoresis (FIGE) for a broad range of the forward pu lse 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 vel ocity showed a sharp positive peak; a similar spike, but with negative velocity, occurred immediately after the field changed from + to -. F or long pulses, the magnitude of this spike increased with M(0.36), re aching 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-stat e plateau. The duration of the velocity trough, and the time of the sm all peak, increased as M(1). For standard FIGE conditions (ratio of fo rward:reverse pulse duration, T-+:T = 3:1; equal forward and reverse f ield amplitudes, E(+) = E(-)), the mobility mu = integral vdt over a c omplete cycle was a minimum when E(+) terminated at the end of the vel ocity trough. The minimum occurred because the velocity during E(+) sa mpled 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 simul ations of FIGE by Zimm (J. Chem. Phys. 1991, 94, 2187-2206) and by Duk e and Viovy (J. Chem. Phys. 1992, 96, 8552-8563) generate real-time ve locities that are in excellent agreement with our experimental data.