Ethidium bromide-mediated renaturation of denatured closed circular DNAs: mechanistic aspects and fractionation of DNA on a molecular weight basis

Abstract

When closed circular duplex DNAs are exposed to alkali in the presence of ethidium bromide, from 0 to 100% of the DNA can be recovered as the fully based-paired duplex (native) form upon neutralization of the solutions. The fraction of native DNA depends on the concentration of ethidium bromide, time of incubation, ionic strength and temperature of the solutions before neutralization as well as the molecular weight and superhelix density of the DNA. Limiting ethidium concentrations exist below and above which 0 and 100% of the DNA, respectively, is recovered as native material under a given set of incubation conditions regardless of the length of time of incubation before neutralization. The strong molecular weight dependence of the fraction of DNA recovered in the native form after a given time of pre-neutralization incubation at ethidium concentrations between the limiting values noted above allows larger DNAs to remain fully denatured upon neutralization while smaller DNAs in the same mixture are fully renatured. This permits the rapid fractionation of mixtures of closed duplex DNAs on the basis of molecular weight when a technique for the separation of denatured from fully base-paired DNA is applied to such mixtures. Such a separation has been demonstrated through the marked enrichment of plasmid cloning vector DNA containing cloned inserts in the fractions that remain denatured after neutralization of alkaline solutions of these DNAs containing ethidium bromide.