Fractionation of mouse DNA in preparative Ag⁺-Cs₂SO₄ gradients

The behavior of nuclear mouse DNA in preparative Ag⁺-Cs₂SO₄ gradients was investigated. Complexing DNA with Ag⁺ at pH 8.8 resulted in a marked increase in mainband buoyant density, unaccompanied by an increase in satellite DNA buoyant density at low molar ratios of Ag⁺ to DNA-P. At higher molar ratios the buoyant density of satellite DNA increased more rapidly than that of mainband DNA so that the two DNA species eventually became inseparable. A plot of buoyant density (ρ{variant}) versus the molar ratio of AG⁺ to DNA-P (R_f) allowed determination of optimal conditions for separation of mainband DNA from satellite DNA. Combined with density gradient relaxation, the technique allowed separation of milligram quantities of mouse DNA into the two molecular species. The addition of increasing amounts of Ag⁺ also produced a speading of the distribution of mainband DNA (σ), followed by contraction as the amount of silver was further increased. Analysis in neutral CsCl in the analytical ultracentrifuge of samples from various parts of a widely spread distribution showed little, if any, separation on the basis of simple G+C content. Much of the behavior of mouse DNA in these gradients can be explained by postulating two types of silver binding sites: a high affinity site which is absent or present in reduced amount in satellite DNA and a low affinity site which is present in both species. It is suggested that these sites may be related to nucleotide sequence(s).