High-performance liquid chromatography of DNA restriction fragments

Abstract

High-performance liquid chromatography on Nucleogen-DEAE 4000-10 has been applied to several problems of the isolation of DNA restriction fragments. Large amounts of DNA fragments of high purity are necessary for biophysical studies and for molecular hybridization in basic research, as well as in medical diagnosis. The influence of various parameters, such as buffer, pH, eluting salt, gradient slope, flow-rate and the addition of urea on the resolution of fragments by high-performance liquid chromatography were studied on an analytical scale, and the optimal conditions were then used for the large-scale preparation of milligram amounts. The best resolution of fragments between 25 and 1500 base pairs was obtained with a linear gradient from 500 mM to 1200 mM sodium chloride in 6 M urea -30 mM sodium phosphate (pH 6.0). Quantitative data are given for the purity and recovery of the sample, and the capacity and lifetime of the column. The following applications of high-performance liquid chromatography of restriction fragments are described: preparation of 2 mg of fragments, separation of 1 mg of DNA insert from 7 mg of its plasmid vector, and analysis of DNA-RNA hybrids.