Preparative Concentration and Size Fractionation of DNA by Porous Media Using a Combination of Flow and Low Electric Field Strength
Preparative Concentration and Size Fractionation of DNA by Porous Media Using a Combination of Flow and Low Electric Field Strength
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DOI：
10.1021/bp970025a
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发表时间：
1997
影响因子：
2.9
通讯作者：
K. Cole
中科院分区：
文献类型：
--
作者：
K. Cole
研究了在外加轴向电场作用下，多孔介质在色谱中对DNA的保留。在填充了Sephadex G‐75和G‐25的柱中，通过负电场保留DNA。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（正极在柱入口）。正电场并不能有效地保留DNA。保留DNA所需的电场强度取决于缓冲液的流速。使用具有更高交联度的凝胶过滤介质Sephadex G‐25来保留DNA，需要比更多孔的Sephadex G‐75更高的电场。在色谱床的入口用电场将稀释的DNA溶液浓缩。DNA限制片段的混合物用于测定大小依赖性保留。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，低分子量DNA片段的保留程度不如高分子量DNA片段。降低流速或增加电场强度会增加低分子量DNA片段的保留率。这样，通过选择一组特定的条件（包装材料、流速和电场强度），可以调节色谱柱保留的DNA片段的分子量。在Sephadex G‐75填充的色谱柱中，使用2 V/cm的电场，证明了高分子量DNA从牛血清白蛋白（一种具有高电泳迁移率的蛋白质）中有效分离。
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 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.