Separations of open-circular DNA using pulsed-field electrophoresis

Abstract

The effect of high electric fields on the gel-electrophoretic mobility of open-circular DNA in agarose differs dramatically from that on linear molecules of the same molecular weight. At high fields, sufficiently large circular forms are prevented from migrating into the gel whereas linear molecules and smaller circular DNAs migrate normally. This effect is strongly field dependent, affecting circular molecules of decreasing size with increasing field strength. We have studied this effect with a series of plasmid DNAs ranging from 2.9 to 56 kilobase pairs using continuous and reversing-pulse electric fields. Application of reversing pulses abolishes the effect under certain conditions and supports the model for the gel electrophoresis of open-circular DNA where circular forms are trapped by engaging the free end of an agarose gel fiber.