Preparative concentration and size fractionation of DNA by porous media using a combination of flow and low electric field strength

Biotechnology Progress
K D Cole

Abstract

The retention of DNA by porous media during chromatography with an applied axial electric field was investigated. DNA was retained by negative electric fields in columns packed with Sephadex G-75 and G-25. A negative field was defined as the electric field orientation in which the direction of electrophoresis was opposing the direction of buffer flow (positive electrode at the column inlet). A positive field was not effective at retaining the DNA. The electric field strength required to retain the DNA was dependent upon the buffer flow rate. The retention of DNA using Sephadex G-25, a gel filtration medium with a higher degree of cross-linking, required higher electric fields than the more porous Sephadex G-75. A dilute DNA solution was concentrated at the inlet of the chromatography bed by an electric field. Mixtures of DNA restriction fragments were used to determine size-dependent retention. DNA was size-fractionated by varying the electric field strength and flow rate. At a given electric field strength and flow rate, the lower molecular weight DNA fragments were not as strongly retained as the higher molecular weight fragments. Decreasing the flow rate or increasing the electric field strength resulted in increased retenti...Continue Reading

Citations

May 1, 2004·Annual Review of Physical Chemistry·Edward S Yeung
Aug 15, 2000·Biotechnology and Bioengineering·C Keim, M Ladisch

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