Restricted uptake of ethidium bromide and propidium diiodide by denatured closed circular DNA in buoyant cesium chloride

Abstract

Alkali-denatured closed circular DNA forms, on neutralization, a relatively stable species first described by Pouwels et al. (1968). In contrast to single-stranded DNA, this denatured two-stranded closed circular DNA species bands densely and co-bands approximately with closed circular duplex DNA in ethidium bromide-CsCl equilibrium density gradients. In CsCl gradients containing propidium diiodide, denDNA I is denser than DNA I, nicked circular DNA and single-stranded φX174 viral DNA. The magnitude of the separations between the above DNAs allows preparative isolation of each when all four are present in the same gradient. The denDNA I has a novel open circular appearance in the electron microscope when cast on standard aqueous hypophases. This species becomes tightly twisted when cast on either aqueous or formamide hypophases containing ethidium bromide. We have concluded from these observations that the high buoyant density of denDNA I in dye-CsCl gradients, relative to single-stranded DNA, is the result of a restricted uptake of dye.