High-Speed Separation of Linear and Supercoiled DNA by Capillary Electrophoresis. Buffer, Entangling Polymer, and Electric Field Effects

Abstract

Capillary electrophoresis in dilute and semidilute (slightly entangled) hydroxyethyl cellulose (HEC) is shown to separate linear double-stranded DNA (ds-DNA) and supercoiled plasmid DNA in the size range 1-16 thousand base pairs in 3 min. The mobilities of linear ds-DNA fragments are stronger functions of electric field strength and buffer concentration than the mobilities of supercoiled plasmids. The effects of HEC concentration and molecular weight are similar for both forms of DNA. The behavioral differences, which are attributed to the greater stiffness of the plasmids, can be used to define conditions that maximize resolution of supercoiled and linear ds-DNA of the same or similar number of base pairs.