Capillary Electrophoretic Separation of DNA Restriction Fragments in Mixtures of Low- and High-Molecular-Weight Hydroxyethylcellulose

Abstract

Previous studies (Barron et al., 1993, 1994) have shown that dilute aqueous solutions of hydroxyethylcellulose (HEC) polymers provide an excellent alternative to gel-based DNA separation media for capillary electrophoresis (CE) of DNA restriction fragments (72−23 130 base pairs). DNA separation by CE in HEC solutions is strongly influenced by the average HEC molecular weight as well as by the HEC concentration in the electrophoresis buffer. Here we describe a systematic investigation of the effects of a mixture of low- and high-molecular weight HEC polymers, over a range of concentrations, to form a DNA separation medium with a broad range of polymer chain lengths. Our results show that, relative to separation media containing solely low-molecular-weight or high-molecular-weight HEC polymers, the mixed polymer solutions provide superior separation over the DNA size range of interest, while providing a five-fold viscosity reduction. The addition of a very small amount of high-molecular-weight HEC to a solution of low-molecular-weight HEC leads to a significant improvement in the separation of larger DNA fragments (>603 base pairs), while retaining resolution of smaller DNA fragments. The consequences of these results on the proposed mechanism of DNA separation in un-cross-linked polymer solutions are discussed.