High-frequency alternating-crossed-field gel electrophoresis with neutral or slightly charged interpenetrating networks to improve DNA separation

Toward improving DNA separations, this work reports the effects of high-fre quency square-wave AC fields superimposed perpendicular to the direct curre nt (DC) separation field on DNA migration in both polyacrylamide-based inte rpenetrating networks (IPNs) and in agarose networks. Compared to standard polyacrylamide gels, IPNs allow the separation of larger DNA (9000 bp vs. 5 000 bp at 5 V/cm). In novel polyacrylamide-based IPNs, an alternating curre nt (AC) field of 5 Hz increased the maximum DNA size separable. This effect was extended to larger DNA sizes with increasing electric-field strength u p to and apparently beyond the power supply-limited maximum electric-field strength of 48 V/cm. The orthogonal AC field also increased mobility. These two results combine to yield a reduction in separation time of up to a fac tor of 20 in novel polyacrylamide-based IPNs. When negatively charged acryl ic-acid groups were incorporated into the IPNs, the use of the AC field cha nged the DNA-network interaction, which altered the size dependence of DNA mobility. In agarose gels, an AC field of 50 Hz increased the size range se parable; however, there was no increase in DNA mobility. There was no chang e in size dependence of mobility in an AC field when the number of charged groups in the agarose network was increased. Based on results in the litera ture, possible mechanisms were examined for the effects of the AC field on DNA separation.