Separation of different physical forms of plasmid DNA using a combination of low electric field strength and flow in porous media: effect of different field gradients and porosity of the media.
The retention of different physical forms of DNA by an electric field in a chromatography system was studied. We were able to effectively separate the supercoiled and the open circular forms of plasmid DNA using this type of electrochromatography system. Chromatography columns were packed with porous beads, and an axial electric field was applied so that convective buffer flow opposed the direction of electrophoresis of the DNA. A model system composed of approximately equal amounts of the super-coiled and open circular forms of the plasmid pBR 322 (4322 base pairs) was used to test the separation. Chromatography beads (agarose-based) with different porosities were used to determine the effect of the stationary phase on the separation. The porous media did not have a major effect on the separation, but the best separations were obtained using porous chromatography media made with the highest agarose concentration (10% agarose). Selective elution of plasmid DNA with different forms was obtained by either increasing the flow rates or decreasing the electric field strength (by steps or a gradient). In all the separations, the more compact supercoiled form of the plasmid was retained less strongly than either the open circular form (nicked) or the linear form. High molecular weight host genomic DNA was more strongly retained than the plasmid DNA. Increasing the ionic strength of the buffer improved resolution and capacity. The capacity of the separation was determined by injecting increasing amounts of plasmid DNA. Satisfactory separation was obtained at sample loading of up to 360 microg of total DNA on a column with dimensions of 2.5 by 11 cm (bed volume of 54 mL). The retention of DNA depends upon a counter-current flow of electrophoresis and convective flow and could be regarded as a type of field flow fractionation. The retention of the DNA by the electric field and flow is discussed in relation to the diffusion coefficients of the DNA.
Cole
K D
KD
National Institute of Standards and Technology, Biotechnology Division, Gaithersburg, MD 20899, USA. Kenneth.cole@nist.gov
Tellez
C M
CM
Blakesley
R W
RW
D002021:Buffers; D002845:Chromatography / Q000379:methods*; D004278:DNA, Superhelical / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D008970:Molecular Weight; D009994:Osmolar Concentration; D010957:Plasmids / Q000302:isolation & purification*
D002021:Buffers; D004278:DNA, Superhelical