A 502-Base Free-Solution Electrophoretic DNA Sequencing Method Using End-Attached Wormlike Micelles

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• 作者：Stephen B. Istivan ; Daniel K. Bishop ; Angela L. Jones ; Shane T. Grosser ; James W. Schneider
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：November 17, 2015
• 年：2015
• 卷：87
• 期：22
• 页码：11433-11440
• 全文大小：498K
• ISSN：1520-6882

文摘

We demonstrate that the use of wormlike nonionic micelles as drag-tags in end-labeled free-solution electrophoresis (鈥渕icelle-ELFSE鈥? provides single-base resolution of Sanger sequencing products up to 502 bases in length, a nearly 2-fold improvement over reported ELFSE separations. 鈥淐iEj鈥?running buffers containing 48 mM C12E5, 6 mM C10E5, and 3 M urea (32.5 掳C) form wormlike micelles that provide a drag equivalent to an uncharged DNA fragment with a length (伪) of 509 bases (effective R_h = 27 nm). Runtime in a 40 cm capillary (30 kV) was 35 min for elution of all products down to the 26-base primer. We also show that smaller Triton X-100 micelles give a read length of 103 bases in a 4 min run, so that a combined analysis of the Sanger products using the two buffers in separate capillaries could be completed in 14 min for the full range of lengths. A van Deemter analysis shows that resolution is limited by diffusion-based peak broadening and wall adsorption. Effects of drag-tag polydispersity are not observed, despite the inherent polydispersity of the wormlike micelles. We ascribe this to a stochastic size-sampling process that occurs as micelle size fluctuates rapidly during the runtime. A theoretical model of the process suggests that fluctuations occur with a time scale less than 10 ms, consistent with the monomer exchange process in nonionic micelles. The CiEj buffer has a low viscosity (2.7 cP) and appears to be semidilute in micelle concentration. The large drag-tag size of the CiEj buffers leads to steric segregation of the DNA and tag for short fragments and attendant mobility shifts.