Plasmid Topoisomer Separation by Capillary Gel Electrophoresis

摘要

We report capillary electrophoretic separation of pUC8 and pBr322 plasmid topoisomers in cross-linked polyacrylamide (PAA) gels in 1X TBE buffer. Plasmid topoisomers are supercoiled forms that have exactly the same chain length but differ in their number of superhelical turns. Because the size in base pairs is invariant, topoisomer mobilities reflect conformational details and differ by only small increments. In cross-linked PAA rapid topoisomer separation can be achieved by DC electrophoresis in capillary lengths as short as 3 cm and near-baseline resolution in longer capillaries. We propose that the separation depends upon the regular structure obtained when a gel is prepared intra-capillary. The isothermal environment promotes formation of a cross-linked polymer of low polydispersity. Such PAA is a sieving matrix of high resolving power, but usable over a relatively narrow DNA size range. It is also possible to prepare gels in which a wide base pair range of supercoiled and nicked plasmids as well as linear ds-DNA may be separated, but without topoisomers resolution. In this paper, we discuss the latest results in topoisomer resolution using a range of plasmids employed in molecular biology and gene therapy.