Chromatographic resolution of complementary strands of denatured Pneumococcal DNA.

It has been shown that AMAK columns are capable of resolving the complementary strands of pneumococcal DNA. Using stepwise elution of denatured DNA, two fractions were obtained at different eluting salt molarities, which regained high biological activity on annealing only when previously mixed.' Unfortunately, resolution could not clearly be demonstrated by means of chromatographic profiles themselves, since denatured DNA could be recovered only very poorly with continuous gradient elution. This difference from the behavior of native DNA had previously been described by others.2' 3 Accomplishment of complete strand resolution even by means of stepwise elution from MAK has been hindered by two factors: (1) incomplete recovery of the DNA and (2) variations in the precise salt molarities at which the two fractions appeared, although the same increment in concentration sufficed to resolve them. The uncertainty in the molarity of the eluting salt solution would be of only trivial importance were it possible to recover the denatured DNA with a continuous gradient. The experiments to be described demonstrate that when the columns are maintained at low temperatures, recovery with gradient elution is complete and the denatured pneumococcal DNA is eluted in two peaks. Since these peaks are shown to reflect complementary strand resolution, the chromatographic profiles may now be used as an independent indication of when this is occurring. Materials and Methods.-Preparation of pneumococcal DNA: DNA was prepared by the method usually employed in this laboratory.4 Deproteinization was effected by the chloroform emulsion technique. Alkali denaturation of DNA: Denaturation was accomplished by diluting a concentrated stock DNA solution into 0.10 N NaOH at room temperature. After 5 min at the alkaline pH, the solution was neutralized to pH 6.8 by addition of the appropriate quantity of 1 M NaH2PO4 (two equivs. per equiv. of NaOH). Before adsorption to a MAK column the salt molarity was increased by addition of 5.0M NaCl. The final concentration of DNA was about 50 ,g/ml. MAK chromatography: The preparation of MAK (methylated albumin adsorbed to kieselguhr) was as previously described.3' 4 Gradient elution was accomplished by dropwise addition of a concentrated salt solution to the initial salt solution in a closed mixing chamber attached to the column. All salt solutions contained 0.02 M potassium phosphate buffer at pH 6.8. The molarities and volumes used resulted in nearly linear salt gradients. The usual gradient was achieved by adding 1.50 M buffered NaCl to 500 ml of 0.60 M buffered NaCl. Both the column and mixing chamber were jacketed for temperature control. The gradients indicated by the dashed lines in Figures 1 and 2 refer to the molarity of eluting salt solution entering the column at the time the frac-