Size Fractionation of DNA Fragments Ranging from 20 to 30000 Base Pairs by Liquid/Liquid Chromatography

Abstract

A chromatographic procedure for the size fractionation of DNA fragments ranging from 20 to 30000 base pairs within a single chromatographic run is described. The procedure was developed from a method communicated about two years ago [Müller, W. et al. (1979) Nucleic Acids Res. 7, 2483–2499] which applies the aqueous two‐phase system formed by poly(ethylene glycol) and dextran using cellulose as support for the lower, dextranrich stationary phase. The parameters found to exert a marked influence on the resolution and the capacity of a column include the ratio of mobile phase to bound phase in the column (phase ratio), the temperature, and the ionic strength in the phases. Concerning the support, no better material than microgranular cellulose has yet been found. A slight preference of dA + dT‐rich fragments for the mobile phase manifests itself by elution anomalies comparable in extent to the migration anomalies of the same fragments in polyacrylamide gels. The resolution observed approaches that of polyacrylamide and agarose gels, but the capacity is 200–500 times higher. The DNA fragments passed through the fractionation procedure seem to be fully susceptible to the enzymatic restriction and modification reactions. Copyright © 1982, Wiley Blackwell. All rights reserved