Cole K D
National Institute of Standards and Technology, Biotechnology Division, Gaithersburg, Maryland 20899, USA.
Biotechnol Prog. 1997 May-Jun;13(3):289-95. doi: 10.1021/bp970025a.
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.
研究了在施加轴向电场的色谱过程中多孔介质对DNA的保留情况。在填充有葡聚糖G - 75和G - 25的柱中，DNA被负电场保留。负电场定义为电泳方向与缓冲液流动方向相反的电场方向（柱入口处为正极）。正电场对保留DNA无效。保留DNA所需的电场强度取决于缓冲液流速。使用交联度更高的凝胶过滤介质葡聚糖G - 25保留DNA比孔隙率更高的葡聚糖G - 75需要更高的电场。通过电场在色谱床入口处浓缩稀DNA溶液。使用DNA限制片段混合物来确定大小依赖性保留。通过改变电场强度和流速对DNA进行大小分级。在给定的电场强度和流速下，较低分子量的DNA片段不如较高分子量的片段保留得牢固。降低流速或增加电场强度会导致较低分子量DNA片段的保留增加。通过这种方式，通过选择一组特定条件（填充材料、流速和电场强度），可以调节柱保留的DNA片段的分子量。在填充有葡聚糖G - 75的柱中，使用2 V/cm的电场证明了从具有高电泳迁移率的蛋白质牛血清白蛋白中高效分离高分子量DNA。
