Department of Chemical Engineering and ‡Department of Biomedical Engineering, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.
Anal Chem. 2015 Nov 17;87(22):11433-40. doi: 10.1021/acs.analchem.5b02931. Epub 2015 Nov 2.
We demonstrate that the use of wormlike nonionic micelles as drag-tags in end-labeled free-solution electrophoresis ("micelle-ELFSE") provides single-base resolution of Sanger sequencing products up to 502 bases in length, a nearly 2-fold improvement over reported ELFSE separations. "CiEj" running buffers containing 48 mM C12E5, 6 mM C10E5, and 3 M urea (32.5 °C) form wormlike micelles that provide a drag equivalent to an uncharged DNA fragment with a length (α) of 509 bases (effective Rh = 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.
我们证明，在末端标记自由溶液电泳（“胶束-ELFSE”）中使用蠕虫状非离子胶束作为拖曳标签，可以将桑格测序产物的分辨率提高到 502 个碱基，比报道的 ELFSE 分离提高近 2 倍。在含有 48mM C12E5、6mM C10E5 和 3M 尿素的“CiEj”运行缓冲液（32.5°C）中，形成蠕虫状胶束，其拖曳等效于长度为 509 个碱基的无电荷 DNA 片段（有效 Rh = 27nm）。在 40cm 毛细管（30kV）中运行 35 分钟，可洗脱所有产物，直至 26 个碱基的引物。我们还表明，较小的 Triton X-100 胶束在 4 分钟的运行中可得到 103 个碱基的读长，因此使用两种缓冲液在单独的毛细管中对桑格产物进行组合分析，可以在 14 分钟内完成所有长度的分析。Van Deemter 分析表明，分辨率受到基于扩散的峰展宽和壁吸附的限制。尽管蠕虫状胶束具有固有多分散性，但未观察到拖曳标签多分散性的影响。我们将其归因于在运行过程中胶束大小快速波动时发生的随机尺寸采样过程。该过程的理论模型表明，波动发生的时间尺度小于 10ms，与非离子胶束中的单体交换过程一致。CiEj 缓冲液的粘度低（2.7cP），并且在胶束浓度下似乎呈半稀状态。CiEj 缓冲液的大拖曳标签尺寸导致 DNA 和标签对短片段的空间隔离和伴随的迁移率变化。
