Detection of Partial Denaturation in AT-Rich DNA Fragments by Ion-Pair Reversed-Phase Chromatography

Abstract

Temperature-dependent denaturation of DNA restriction fragments from the pBR322 plasmid ranging in length from 46 to 910 base pairs was detected by ion-pair reversed-phase high-performance liquid chromatography using columns packed with alkylated nonporous poly(styrene/divinylbenzene) particles. The presence of acetonitrile in the mobile phase was found to decrease the melting temperatures of DNA fragments by 1.5-2 °C/% of acetonitrile in the eluent. Small fragments (<120 bp) were completely denatured between 53.6 and 63.5 °C, depending on their total GC content. Whereas retention times of completely helical DNA fragments increased gradually with increasing temperature, partial denaturation of larger DNA fragments (>150 bp) was found to reduce retention at temperatures above 53.6 °C. Therefore, micropreparative fractionation and rechromatography, together with DNA restriction analysis, were applied to identify the correct elution order of completely helical and partially denatured fragments. Inspection of the DNA sequences of partially denatured fragments revealed domains with repeating AT base pairs. Positions of partial denaturation within the pBR322 plasmid detected by chromatographic analysis were in good agreement with partial denaturation maps obtained by electron microscopy desrcibed in the literature.