Free-solution electrophoresis of DNA modified with drag-tags at both ends
Free-solution electrophoresis of DNA modified with drag-tags at both ends
复制标题
DOI：
10.1002/elps.200500554
复制
发表时间：
2006-05-01
期刊：
影响因子：
2.9
通讯作者：
Barron, Annelise E.
中科院分区：
文献类型：
--
作者：
Meagher, Robert J.;McCormick, Laurette C.;Barron, Annelise E.
在末端标记自由溶液电泳（ELFSE）中，DNA分子被标记为摩擦修饰剂或“拖拽标签”，允许它们在自由溶液中进行基于大小的电泳分离。在使用链霉亲和素作为拖标签的dsDNA的早期工作中，有趣的观察结果是，在两端加入链霉亲和素标签所引起的拖阻明显超过单个链霉亲和素的两倍（Heller， C. et al.， J. chrorogr a 1998,806, 113-121）。这一发现被认为是错误的，随后的工作集中在只在DNA分子的一端附加单个拖拽标签的实验上。最近的理论工作（McCormick， L. C., Slater， G. W., Electrophoresis, 2005, 26,1659-1667）研究了末端效应对带电-不带电聚合物共轭物的自由溶液电泳迁移率的贡献，重新提出了在两端放置拖曳标签增强拖曳的问题。在本研究中，实验研究了这种效应，使用定制合成的ssDNA寡核苷酸，允许将拖拽标签附着在一端或两端，以及用引物生成的dsDNA PCR产物，适合将拖拽标签附着在一端或两端。各种大小的拖标签被使用，包括合成多肽拖标签以及基因工程蛋白聚合物拖标签。标记两端所产生的增强阻力已被证实，标记两端所产生的ssDNA的额外阻力为6-9%，dsDNA的额外阻力为10-23%，而不是简单地将一端的拖标签大小增加一倍。对两端标记的ssDNA的实验结果与最近的末端效应理论的预测进行了比较，具有相当好的定量一致性。这些实验结果证明了通过标记DNA分子的两端来增强ELFSE分离的可行性，从而提高了该技术的分辨率和更广泛的应用范围。
In end-labeled free-solution electrophoresis (ELFSE), DNA molecules are labeled with a frictional modifier or "drag-tag", allowing their size-based electrophoretic separation in free solution. Among the interesting observations from early work with dsDNA using streptavidin as a drag-tag was that the drag induced by including a streptavidin label at both ends was significantly more than double that from a single streptavidin (Heller, C. et al., J. Chromatogr A 1998, 806, 113-121). This finding was assumed to be in error, and subsequent work focused on experiments in which only a single drag-tag is appended to one end of the DNA molecule. Recent theoretical work (McCormick, L. C., Slater, G. W., Electrophoresis 2005, 26,1659-1667) has examined the contribution of end-effects to the free-solution electrophoretic mobility of charged-uncharged polymer conjugates, reopening the question of enhanced drag from placing a drag-tag at both ends. In this study, this effect is investigated experimentally, using custom-synthesized ssDNA oligonucleotides allowing the attachment of drag-tags to one or both ends, as well as dsDNA PCR products generated with primers appropriate for the attachment of drag-tags at one or both ends. A range of sizes of drag-tags are used, including synthetic polypeptoid drag-tags as well as genetically engineered protein polymer drag-tags. The enhanced drag arising from labeling both ends has been confirmed, with 6-9% additional drag for the ssDNA and 10-23% additional drag for the dsDNA arising from labeling both ends than would be expected from simply doubling the size of the drag-tag at one end. The experimental results for ssDNA labeled at both ends are compared to the predictions of the recent theory of end-effects, with reasonably good quantitative agreement. These experimental findings demonstrate the feasibility of enhancing ELFSE separations by labeling both ends of the DNA molecule, leading to greater resolving power and a wider range of applications for this technique.