An improved method for the isolation of supercoiled DNA molecules using ion-exchange column chromatography

Abstract

An improved procedure is presented for the rapid isolation of supercoiled molecules using ion-exchange chromatography of alkali-denatured material. Under the conditions employed, contaminating cellular DNA, RNA, and nicked circular molecules are denaturable and are presented to the column as single strands while intact, covalently closed, supercoiled molecules resist denaturation and are batch eluted from the column as the only remaining duplex species. Small amounts of protein and short, duplex fragments (less than 1 kb) are removed in the void volume. There is no need for gradient fractionation. Supercoil fractions are virtually free of contamination with linear molecules as determined by electron microscopy, have an A260 nm/A280 nm ratio approaching 2.0, and are suitable for subsequent heteroduplex analysis, molecular cloning, subcloning, or restriction enzyme mapping. The method has been used successfully to purify plasmids up to 45 kb in length and to isolate rare circular replication intermediates from an overwhelming excess of contaminating linear molecules in retrovirus-infected cells.