Two-dimensional gel electrophoresis of circular DNA topoisomers

This chapter discusses the two-dimensional gel electrophoresis of circular DNA topoisomers. The major problem with one-dimensional gel electrophoresis is the relatively limited range over which migration is a function of topology. Two-dimensional gel electrophoresis increases this range by a considerable degree, and it provides a simple way to reveal topology-dependent structural transitions and measure some parameters for the process. The geometric distortion of a supercoiled molecule means that the average shape of the molecule is changed as a result of its topology and the result is altered frictional properties. This can be done by agarose gel electrophoresis. This shows the result of electrophoresing a series of topoisomers of the 3658-bp plasmid pAT153 in 1% agarose. A ladder of species is observed, each member of which is a single topoisomer differing from its neighbors by a linking difference of ±. The differences in shape among the different topoisomers results in a progressive change in electrophoretic mobility—that is, the more supercoiled the species, the faster the migration. The resolving power of agarose gels for topoisomers is impressive, but the range is limited so that the more highly supercoiled species comigrate as a broad band at the lower end of the gel. The resolution can be improved for smaller DNA molecules by the use of composite polyacrylamide–agarose gels.