Sequencing of DNA by free-solution capillary electrophoresis using a genetically engineered protein polymer drag-tag
Sequencing of DNA by free-solution capillary electrophoresis using a genetically engineered protein polymer drag-tag
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DOI：
10.1021/ac702591t
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发表时间：
2008-04-15
影响因子：
7.4
通讯作者：
Barron, Annelise E.
中科院分区：
文献类型：
--
作者：
Meagher, Robert J.;Won, Jong-In;Barron, Annelise E.
我们展示了首次使用非天然基因工程蛋白质聚合物拖标签通过末端标记自由溶液电泳 (ELFSE) 对 DNA 片段进行测序。通过自由溶液毛细管电泳分离桑格循环测序反应产生的荧光标记的 DNA 片段，与之前报道的该技术相比，具有更高的分辨率和更清晰的结果。利用 ELFSE，在没有筛分基质的情况下，通过端接聚合物“拖标签”实现基于尺寸的 DNA 分离，该聚合物“拖标签”以尺寸依赖的方式改变 DNA 的电荷摩擦比。此前，由于缺乏合适的大型单分散拖标签，ELFSE 分离的进展受到限制。为了解决这个问题，我们设计、构建、克隆、表达和纯化了一种非天然的基因工程 127 聚体蛋白质聚合物，用作 ELFSE 拖动标签。桑格循环测序反应是通过共价连接到测序引物的拖曳标签进行的，这比之前的 ELFSE 测序策略是一个重大进步。电泳分离是扩散限制的，拖曳标签不会明显吸附到毛细管壁上。尽管读长（约 180 个碱基）仍然很短，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动标签可以将 ELFSE 的读长延伸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，而没有任何与粘性聚合物筛网相关的困难，因此适合在微通道和基于芯片的电泳系统中实施。
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 electorophoresis, 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 "dragtag" 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.