Effect of one-dimensional pulsed-field gel electrophoresis on linear and circular DNA.

Abstract

A summary of the three main one-dimensional pulsed-field strategies (zero-integrated field, forward-biased field, and high frequency modulation) used for separating DNA molecules without band inversion within a preselected size range is given. Each of these strategies has size-specific features which make separations up to 6 Mbp possible. We applied the same methodology to circular DNAs varying in size from 2 kbp to about 4 Mbp. The migration of intermediate-sized circular plasmids (50 kbp-400 kbp) under these pulse conditions remains unexplained. On the other hand, preliminary results show that the migration of very large molecules, which are expected to be circular, comigrate with linear chromosomes of the same size under certain pulse conditions. We hypothesize that sample preparation, or the effect of the pulsed field, can create breakage and linearize very large circular DNAs, or that very large circular DNAs (> 2 Mbp) act like linear DNAs of the same size when submitted to one-dimensional pulsed-field gel electrophoresis conditions. The most likely possibility is that some of the circular DNAs have been linearized with one break during sample preparation, giving rise to a band at about 4 Mbp. The circular DNAs with more than one break may form an indistinguishable smear.