On the movement and alignment of DNA during 120 degrees pulsed-field gel electrophoresis.

The displacement per pulse of lambda, T4, and G DNA during pulsed-field agarose gel electrophoresis has been measured for a fine mesh of pulse durations T between 0.02 and 120 s. The slopes of these curves show that the DNA moves by two distinct processes, designated 1 and 2, depending upon the pulse duration T. Process 1 operates at short T and causes dx/dT to decrease gradually with increasing T. This process is independent of molecular weight M. Process 2 is effective at longer T and causes dx/dT to rise sharply in sigmoidal fashion at a value of T which increases as M1.2, finally reaching a plateau of 1.4 microns/s for E = 4 V/cm. The shape of the dx/dT curve and its dependence on M lead directly to 4 zones of separation in plots of mobility vs M for different T. The alignment of the 3 DNAs during PFGE was measured by fluorescence-detected linear dichroism for E between 4 and 10 V/cm. These results are used in developing a molecular understanding of the mobility data.