Meagher Robert J, Won Jong-In, Coyne Jennifer A, Lin Jennifer, Barron Annelise E
Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, USA.
Anal Chem. 2008 Apr 15;80(8):2842-8. doi: 10.1021/ac702591t. Epub 2008 Mar 5.
We demonstrate the first use of a non-natural, genetically engineered protein polymer drag-tag to sequence DNA fragments by end-labeled free-solution electrophoresis (ELFSE). Fluorescently labeled DNA fragments resulting from the Sanger cycle sequencing reaction were separated by free-solution capillary electrophoresis, with much higher resolution and cleaner results than previously reported for this technique. With ELFSE, size-based separation of DNA in the absence of a sieving matrix is enabled by the end-on attachment of a polymeric "drag-tag" that modifies the charge-to-friction ratio of DNA in a size-dependent fashion. Progress in ELFSE separations has previously been limited by the lack of suitable large, monodisperse drag-tags. To address this problem, we designed, constructed, cloned, expressed, and purified a non-natural, genetically engineered 127mer protein polymer for use as an ELFSE drag-tag. The Sanger cycle sequencing reaction is performed with the drag-tag covalently attached to the sequencing primer, a major advance over previous strategies for ELFSE sequencing. The electrophoretic separation is diffusion-limited, without significant adsorption of the drag-tag to capillary walls. Although the read length (at about 180 bases) is still short, our results provide evidence that larger protein polymer drag-tags, currently under development, could extend the read length of ELFSE to more competitive levels. ELFSE offers the possibility of very rapid DNA sequencing separations without any of the difficulties associated with viscous polymeric sieving networks and hence will be amenable to implementation in microchannel and chip-based electrophoresis systems.
我们展示了首次使用非天然的、基因工程改造的蛋白质聚合物拖尾标签，通过末端标记自由溶液电泳（ELFSE）对DNA片段进行测序。桑格循环测序反应产生的荧光标记DNA片段通过自由溶液毛细管电泳进行分离，分辨率比此前报道的该技术更高，结果也更清晰。通过ELFSE，在没有筛分基质的情况下，基于大小的DNA分离是通过聚合物“拖尾标签”的末端连接实现的，该标签以大小依赖的方式改变DNA的电荷与摩擦比。此前，ELFSE分离的进展一直受到缺乏合适的大尺寸、单分散拖尾标签的限制。为了解决这个问题，我们设计、构建、克隆、表达并纯化了一种非天然的、基因工程改造的127聚体蛋白质聚合物，用作ELFSE拖尾标签。桑格循环测序反应是在拖尾标签共价连接到测序引物的情况下进行的，这是ELFSE测序先前策略的一个重大进展。电泳分离受扩散限制，拖尾标签不会显著吸附到毛细管壁上。尽管读取长度（约180个碱基）仍然较短，但我们的结果提供了证据，表明目前正在开发的更大的蛋白质聚合物拖尾标签可以将ELFSE的读取长度扩展到更具竞争力的水平。ELFSE提供了非常快速的DNA测序分离的可能性，而没有与粘性聚合物筛分网络相关的任何困难，因此将适用于微通道和基于芯片的电泳系统。
