Separation of Nucleotides, Nucuosides and Bases of DNA with Cation Exchange Columns

Abstract

Dovex-50-X4 of each 400 was used after charge reduction for the separation of DNA nucleotides or nucleosides or bases or mixtures of these. The method is simple. A 50 al burst was plugged with glass wool and is packed with the resin at cold running water aspirator pressure and prewashed with 250 al of the elation buffer. In general, the molarity and the pH of the prewash buffer were the same as those of the first elution buffer. The sample mixtures containing 1 mg of each of the compounds ware dissolved in 0.5 ml of the elation buffer or 0.1 N HCl and then passed through the column and eluted at the approximate rate of 1 ml/8 min at room teaperature under gravity flow (atmospheric pressure). The nucleotides were eluted by 0.05 M ammonium format buffer pH 3.2 while the nucleosides and the bases ware elutad by 1.0 M ammonium format buffer of pH 4.0 mad 3.5 respectively. The mixed nucleosides and nucleotides ware eluted in a stepwise manner by 0.02 M ammonium formats buffer pH 3.2 followed by 1 M ammonium formate buffer pM 4.0 whereas the mixed nucleosides and baaes were eluted by 1 M ammonium formate buffer pH 3.8. A mixture of thymine, thymidine, and thymidylic acid was eluted by 0.05 M ammonium formate buffer pH 3.2. The eluatas were collected using a fraction collector and read on a Beckman DU spectrophotometer. The procedure gives between 90 and 95% recovery of the compounds and is also applicable for the separation and purification of nucleosides obtained by eazymatic digests of DNA.