Two-dimensional motion of DNA bands during 120 degrees pulsed-field gel electrophoresis.

The position and velocity of a band of double-stranded, linear DNA from bacteriophage G were measured during 120 degrees pulsed-field gel electrophoresis, using a video micrometer. Both the x and y coordinates were determined simultaneously in the plane of a 1% agarose gel; x is the mean drift direction. For pulse durations T greater than the tube renewal time T*, the path traced by the band of 670 kb DNA in the xy plane was in remarkably good accord with that predicted by Southern's ratchet model. However, the measured instantaneous velocity vx showed a sharp backward spike each time the field changed direction, with amplitude about twice the mean drift velocity. This spike is not consistent with models which assume a constant curvilinear velocity of DNA in a tube, nor with the biased reptation model without fluctuations. The corresponding measurements of vy showed a sharp positive spike with amplitude more than 3 times the plateau velocity in the y direction; neither model predicted this. The sharp velocity spikes are consistent with the idea that, for T > T*, a large fraction of the DNA chains are stretched into U-shaped or herniated configurations. When the field changes direction, the arms of the U's and the hernias recoil rapidly in response to intramolecular DNA chain tension. Because the base of a U or hernia is fixed by gel fibers, the center of mass of the chain recoils backward every time the field changes direction.