Fractionation of mammalian DNA on deae-cellulose.

Abstract

Chromatography on a DEAE-cellulose (DE-52) column of native and denatured DNA from P388F cells has been studied. The main bulk of native DNA is eluted at 0.8M NaC1 and a minor fraction is eluted with 0.5N NaOH. The proportion of the DNA components obtained depends on the type of isotopic labelling used and the method of storing of the DNA preparation following isolation. Heat-denatured DNA elutes in 2 M NaC1 mainly within the pH gradient form 0.1 M NH4OH. When native DNA is chromatographed on DEAE-cellulose, a small fraction of the DNA elutes at 0.5 N NaOH. This fraction is double-stranded, as determined by hydroxyapatite chromatography. A similar component predominates, however, when "newly synthesised" DNA is fracionated. The profiles obtained with "newly synthesised" and "template" labelled DNA differ in their undenatured and heat-denatured configurations. The presence of formaldehyde during the chromatography of undenatured DNA leads to an increased homogeneity of the profile and during heat denaturation considerable modifications to the profile are observed. Some of the changes can be explained in terms of a decrease in the heterogeneity of the charge distribution on the DNA. The technique appears to combine a high degree of reproducibility with sensitivity to charge clustering along the DNA.