An evaluation of methods for the purification of DNA preparations

Four tritiated thymidine-labeled DNA preparations known to contain areas of single strandedness were chromatographed on BNDC, HAP, and MAK. With an ethidium bromide fluorescence technique, the proportion of single-stranded regions in each of the stock preparations and DNA column eluates was evaluated. Although all column methods removed a significant proportion of test material containing single-stranded DNA, only MAK columns consistently yielded homogeneous double-stranded DNA preparations. Variable results were obtained, depending on the preparation chromatographed and the method used. Millipore filtration, although reducing single-stranded contamination, also failed to produce uniformly double-stranded DNA. Serum anti-DNA bindings were measured by the Millipore filter technique with two DNA preparations before and after chromatography. A significant decrease in percent binding was demonstrated with DNA samples fractionated on MAK columns as a result of the removal of most single-stranded DNA regions. Only select sera tested against BNDC and HAP DNA eluates showed a decrease in anti-DNA binding. It is concluded that methods of column chromatography are variably effective in reducing the proportion of single-stranded DNA in mixed DNA preparations. Confirmation of the homogeneous nature of the DNA antigen by structural analysis is recommended.