Newly synthesized mammalian cell DNA. Separation of Okazaki pieces by thermal chromatography on hydroxyapatite

The optical melting profile of a fraction of nascent DNA which can be isolated from DNA of logarithmically growing Ehrlich ascites cells by nitrocellullose column chromatography was recorded. In relation to the bulk DNA a slightly lowered Tm and an increased width of the transition was observed. Thermal hydroxyapatite chromatography revealed that melting of the nascent DNA begins at relatively low temperatures in helix regions containing small daughter strand pieces. The pieces were characterized as Okazaki pieces by determining their size and their labeling kinetics during [3H] thymidine pulses. The combination of nitrocellulose chromatography and thermal hydroxyapatite chromatography seems to be suitable for isolating Okazaki pieces.