Fractionation of DNA on Hydroxyapatite with a Base-Specific Complexing Agent

Abstract

We describe a chromatographic technique for separating mixtures of DNA of varying G + C content. The method employs a specially prepared high-capacity hydroxyapatite and a G·C-specific DNA ligand (2-methyl-3-amino-7-dimethylamino-5-phenyl-phenazinium cation, abbreviated PNR). DNA molecules rich in G·C pairs bind larger amounts of this dye and are eluted earlier from the hydroxyapatite column than are molecules rich in A·T pairs. The dye can easily be removed from DNA by dialysis or solvent extraction after the chromatographic separation. The resolution of the method approaches that of CsCl density gradient separation, and the capacity of the column is much larger than that of a typical density gradient experiment. Elution of the DNA is not dependent on molecular weight, so samples of different molecular weight can be separated on the basis of G + C content. The technique should be especially useful for separating G·C-rich minor components from DNA obtained form eukaryotic cells, as demonstrated by a fraction of DNA from calf thymus.