Elastic Bag Model of One-Dimensional Pulsed-Field Gel Electrophoresis (ODPFGE)

Abstract

Gel electrophoresis is one of the most common techniques used in molecular biology for the separation of DNA molecules. Conventional gel electrophoresis (using a static electric field) does not permit separation of DNA fragments larger than 30-50 kbp (1) as shown in Fig. 1A of Chapter 7 . This is a surprising result as one would think that larger molecules would suffer a larger retardation, and separation over any size range would be possible. The inability to separate is related to the molecular conformation of a polyelectrolyte, such as DNA, migrating in a disordered medium, such as a gel, under the influence of a static electric field. During continuous field electrophoresis, the larger DNA fragments tend to orient and stretch in the field direction because they migrate in a one-dimensional fashion between the gel fibers (2-4). When this orientation is negligible, e.g., for smaller molecules or for very low field intensities, they maintain a three-dimensional random-walk conformation intertwined with the gel fibers during migration, and experience a retardation that is proportional to the mol size. However, when the orientation becomes large, the molecules become stretched and migrate essentially linearly along the field direction (5). The electrophoretic mobility then becomes independent of the mol size and no separation of molecules of different sizes is possible (Fig. 1A of Chapter 7 ). Physically, this is a consequence of the fact that for long molecules stretched and oriented in the field direction, both the electrical force on the molecule and the average friction opposing the forward motion are proportional to the length. It follows that the velocity, which is the ratio of these two quantities, depends only on the force per unit length and is independent of the actual mol length. This explains why a plateau of length-independent mobility is reached in a continuous electric field (Fig. 2 of Chapter 7 ).