Chromatography of deoxyribonucleic acids on calcium phosphate columns

Abstract

Calf thymus DNA in 0.005 M phosphate buffer (pH 6.7) was subjected to Chromatographic analysis on calcium phosphate columns in conjunction with linear gradient elution with phosphate solution ranging from 0.005 to 0.4 M at constant pH. Eluant fractions (3–5 ml.) were collected hourly for 120 hr. The course of elution of DNA was followed by measuring the absorbance of each fraction at 260 mμ. It was found that DNA initially adsorbed on the column was not eluted until the phosphate concentration of the eluting solution approached 0.15 M. Up to 72% of total ultraviolet-absorbing material was eluted in the range between 0.15 and 0.25 M phosphate. No additional elution peaks appeared between 0.25 and 0.40 M phosphate. The column was evaluated with regard to its capacity to differentiate DNA from the products obtained by short-term (30-min.) digestion of DNA by DNAse. Chromatographic analyses of mixtures of untreated DNA with such DNA digests revealed at least three major fractions as indicated by distinct elution peaks. The first fraction was not adsorbed by the column and came through immediately in 0.005 M phosphate. A second peak appeared at 0.045 M phosphate; a third elution maximum occurred at 0.2 M phosphate. The latter peak corresponded to that obtained with untreated DNA when chromatographed alone. The present results imply that high-molecular-weight deoxypoly-nucleotides can be separated from undegraded DNA, under mild conditions of pH and ionic strength, by calcium phosphate column chromatography.