The mechanism of DNA's fractionation during pulsed-field agarose gel electrophoresis: a hypothesis.

The resolution by length of linear double-stranded DNA longer than 10-20 kb is increased by periodically changing direction of the electrical field during agarose gel electrophoresis (pulsed field gel, or PFG, electrophoresis). Previously proposed mechanisms for this effect include viscoelastic relaxation of the DNA and DNA length-dependent path lengths through the gel. Several data are not explained by these mechanisms. To better explain the data, here is proposed: (a) the existence of flexible projections from the more rigid fibers that form the network of agarose gels; these projections slow DNA's motion by threading loops of DNA; (b) the bending of these projections in the direction of DNA's motion during electrophoresis; (c) hysteresis in the re-orientation of these projections when the direction of the electrical field is changed, and (d) increase in resistance to DNA's motion in a direction opposite to that of the projections' bending. By use of (a)-(d), the facts known about DNA fractionation during PFG electrophoresis are qualitatively explained.