AbstractThe migration of a series of supercoiled plasmids ranging in size from 4 to 91 kilobases (kb) has been analyzed by orthogonal‐field‐alternation gel electrophoresis (OFAGE). These circular DNAs enter OFAGE gels and are resolved over the same region of the gel as linear DNAs from 260 to 2200 kb. Furthermore, a distinct triphasic migration pattern was observed for the supercoiled DNAs. The migration of plasmids between 6 and 20, and 60 and 91 kb is inversely proportional to size, whereas the mobilities of plasmids between 20 and 60 kb increase with size. Unlike linear DNA molecules, the relative mobilities of these plasmids are constant over a broad range of pulse times, from 10 to 120 s. Electrophoresis of supercoiled, relaxed, and nicked open circular forms as well as topoisomers of small plasmids shows that the extent of supercoiling has a dramatic effect on plasmid migration on OFAGE. Several practical applications for exploiting the different migration properties of circular and linear DNA molecules on OFAGE are presen