A method is described for the preparative purification of supercoiled DNA of bacterial cell extracts without an ultracentrifugal step. DNA of crude cell lysates, containing supercoiled plasmid DNA and chromosomal DNA fragments is freed of protein by chromatography on Sepharose B4. The separation of closed-circular supercoiled DNA from linear and open-circular DNA is performed by chromatography on hydroxyapatite in the presence of ethidium bromide by taking advantage of the different binding ability of the dye for linear and closed-circular DNA. The best separations are obtained when the linear or nicked DNA in the sample is complexed at a frequency of one dye molecule per three-five base-pairs. The extent of separation seems to depend only on the intrinsic superhelical density of the supercoiled DNA molecules and not on their sizes.
The isolation of supercoiled circular duplex DNA is performed in general by density gradient centrifugation in the presence of the intercalating dye ethidium bromide (1). The separation from linear duplex DNA or nicked-circular DNA by this technique is based on the fact that the supercoiled DNA has a restricted capacity of binding the dye when more than one dye molecule per about ten base-pairs are bound, due to the formation of supercoils with opposite directions of winding. The limited uptake of dye molecules leads to a smaller change in buoyant density of the supercoiled than of the linear DNA. The two DNA species can thus be separated in a density gradient.
Recently we described a method for separating mixtures of DNA of varying G + C content on hydroxyapatite in the presence of a GCspecific intercalating DNA ligand (2). We concluded that the separation is based on the structural change produced in the DNA backbone by the intercalator. This led us to the assumption that supercoiled circular duplex DNA can be separated from linear duplex or nicked-circular DNA by the same technique by taking advantage of the different binding capacities of the two species. The results communicated in this paper 372