Fractionation of DNA restriction fragments with ion‐exchangers for high‐performance liquid chromatography

Abstract

The fractionation abilities of several ion-exchangers of the high-performance liquid chromatography type for two sets of DNA restriction fragments, ranging from 7 base pairs (bp) to about 650 bp and differing in their mean base composition, have been studied. The ion-exchangers tested comprise the RPC-5, the 5-PW DEAE and the Mono Q as polymer-based resins, and the Nucleogens 500 and 4000, both prepared from silica beads. The results indicate that all the ion-exchangers except the 5-PW DEAE perfectly separate fragment sizes up to about 90 bp, the 5-PW DEAE separating to 45 bp only. Above 200 bp only the Mono Q resin works in a satisfactory way provided that about 100 micrograms DNA mixture, containing less than 25 fragments within the given size range, is loaded per milliliter of packed resin. Appreciable base-pair specificities were detected for most of the resins which cause substantial retardations of the d(A + T)-rich fragments with respect to the eluting salt concentration. If the latter dominate in the DNA sample, acceptable results were only obtained with the Mono Q resin when the column was operated at elevated temperature.