Open AccessFractionation of DNA restriction fragments with ion‐exchangers for high‐performance liquid chromatography
Author(s) -
MÜLLER Werner
Publication year - 1986
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1986.tb09478.x
Subject(s) - chromatography , fractionation , ion , chemistry , base (topology) , ion exchange resin , dna , polymer , ion exchange , analytical chemistry (journal) , salt (chemistry) , biochemistry , organic chemistry , mathematical analysis , mathematics
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 μg 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.