Chromatographic fractionation of bacterial deoxyribonucleic acid

1. 1. DNA mixtures were fractionated chromatographically on columns of a polycarboxylate resin (IRC-50). The samples were adsorbed to the resin in magnesium acetate solutions and eluted by the application of continuous gradients of salt concentration. 2. 2. A study was made of the effect of various conditions upon the resolution of artificial mixtures of DNA from different bacterial species. Although elution occurs in increasing concentrations of magnesium acetate, resolving power is greater in gradients of increasing concentration of sodium acetate in constant levels of magnesium acetate or in gradients of decreasing concentration of magnesium acetate. The resolving power was little affected by altering the pH, the column temperature or column height, or by the substitution of Ca²⁺ for Mg²⁺ or of K⁺ for Na²⁺. Resin particle size was found to have a marked influence on resolving power, finer-grade resin yielding higher resolution. 3. 3. Chromatography on the IRC-50 column as well as on the methylated-albumin-kieselguhr column of Mandell and Hershey³ was found to be dependent upon the size and base composition of the molecules. Elution from the IRC-50 column was more sensitive to differences in base composition and that from the methylated-albumin column more sensitive to differences in size. 4. 4. DNA from Micrococcus lysodeikticus was completely resolved from pneumococcal and Escherichia coli DNA, the latter two being eluted later in one peak with E. coli DNA at the heading edge. High-molecular-weight RNA and heat-denatured DNA are not retarded by the column. 5. 5. DNA eluted from the column appeared on rechromatography to have been fractionated with respect to chromatographic properties without denaturation.