Meagher Robert J, McCormick Laurette C, Haynes Russell D, Won Jong-In, Lin Jennifer S, Slater Gary W, Barron Annelise E
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL 60208, USA.
Electrophoresis. 2006 May;27(9):1702-12. doi: 10.1002/elps.200500554.
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.
在末端标记自由溶液电泳（ELFSE）中，DNA分子用摩擦修饰剂或“拖尾标签”进行标记，从而使其能够在自由溶液中基于大小进行电泳分离。在早期使用链霉亲和素作为拖尾标签对双链DNA进行研究时，有一个有趣的发现是，两端都加上链霉亲和素标签所产生的拖曳力明显超过单个链霉亲和素所产生拖曳力的两倍（Heller, C.等人，《色谱杂志A》，1998年，806卷，113 - 121页）。这一发现被认为是错误的，随后的研究工作集中在仅在DNA分子一端附加单个拖尾标签的实验上。最近的理论研究（McCormick, L. C., Slater, G. W.,《电泳》，2005年，26卷，1659 - 1667页）探讨了末端效应对带电 - 不带电聚合物共轭物自由溶液电泳迁移率的影响，重新开启了关于两端都放置拖尾标签会增强拖曳力的问题。在本研究中，使用定制合成的单链DNA寡核苷酸来实验研究这种效应，这些寡核苷酸允许在一端或两端连接拖尾标签，同时还使用了用适合在一端或两端连接拖尾标签的引物生成的双链DNA PCR产物。使用了一系列大小的拖尾标签，包括合成聚肽拖尾标签以及基因工程蛋白聚合物拖尾标签。两端标记所产生的增强拖曳力已得到证实，对于单链DNA，两端标记所产生的额外拖曳力为6 - 9%，对于双链DNA，额外拖曳力为10 - 23%，这比仅将一端拖尾标签大小加倍所预期的要高。将两端标记的单链DNA的实验结果与最近的末端效应理论预测进行了比较，在定量上有相当好的一致性。这些实验结果证明了通过标记DNA分子两端来增强ELFSE分离的可行性，从而为该技术带来更大的分辨能力和更广泛的应用范围。
