Dynamics of DNA under pulsed-field gel electrophoresis as analyzed from birefringence rise and decay.

The orientation of DNA induced by electrophoretic transport in agarose gel has been studied by optical birefringence. From its field-free decay, it is clearly demonstrated that the degree of orientation results from two processes: alignment along the electric field (stretching of the end-to-end vector) and elongation of the primitive path in the gel (overstretching). Separation of the two contributions allows the experimental determination of the effective charge per base pair, the gel pore size seen by the reptating molecule, the reptation time, the degree of overstretching and the mean relaxation time of the overstretching. Their field and DNA length dependences compare well with theoretical predictions. Similarly, the time at which overstretching presents an overshoot in the rise of the orientation follows closely the predictions of a model based on the evolution of J-shaped conformations. The recovery of such conformations is studied by a sequence of two pulses with variable delay time. The use of directly measured or extrapolated characteristic times and fields in the design of efficient pulse schemes for pulsed-field gel electrophoresis is emphasized.