Preparative Concentration and Size Fractionation of DNA by Porous Media Using a Combination of Flow and Low Electric Field Strength

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 retention of the lower molecular weight DNA fragments. In this manner, by selecting a specific set of conditions (packing material, flow rate, and electric field strength), the molecular weight of DNA fragments retained by the column can be adjusted. Efficient separation of high molecular weight DNA from bovine serum albumin, a protein with high electrophoretic mobility, was demonstrated using a field of 2 V/cm in a column packed with Sephadex G-75.

Preparative electrochromatography of proteins in various types of porous media

“…Operation of the EC systems has been described previously [7]. Briefly, water-jacketed columns (1.5´30 cm and 2.5´60 cm) were packed with the appropriate sizeexclusion resin.…”

“…Cole [7] had previously observed that lower electric fields were needed to retain DNA in an EC column as the gel porosity increased. We have now confirmed the same trend in the case of proteins.…”

“…EC dependent on D/m ratios would explain the large difference in electric fields necessary to retain DNA and proteins because their ratios differ significantly (Table 1). Cole [7] reported that electric fields of about 1 V/cm were enough to immobilize l-DNA in an EC column. Here, we needed fields above 20 V/cm to retain BSA and the immunoglobulins were not retained, only retarded, with fields of 30 V/cm.…”

“…This approach would also be able to explain the effect of the flow rate on column capacity. Cole [7] reported that column capacity is an inverse function of flow rate and that the electric field needed to retain DNA was proportional to the flow velocity (although not linearly). An increase in flow rate would reduce the zone of low flow around the bead surface and the distance that a protein must diffuse to be carried away by convection.…”

“…Neremberg and Pogojeff [4] were first to describe a system combining both electrophoresis and flow chromatography for the separation of serum proteins. EC has been employed, over the years, for the separation of several charged biomolecules ranging from hepatitis B antigen [5], to human plasma [6], to DNA fragments [7]. With the introduction of high voltage in capillary columns for analytical separations [8], substrates of even a more diverse nature have been purified [9,10].…”