Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes
Ultrafast DNA sequencing on a microchip by a hybrid separation mechanism that gives 600 bases in 6.5 minutes
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DOI：
10.1073/pnas.0705093105
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发表时间：
2008-01-15
影响因子：
11.1
通讯作者：
Barron, Annelise E.
中科院分区：
文献类型：
--
作者：
Fredlake, Christopher P.;Hert, Daniel G.;Barron, Annelise E.
在人类第二个基因组（Venter’s）完成后，要实现大规模基因组测序的巨大潜力，必须大幅降低其他基因组完整测序的成本。在降低测序成本的技术中，微芯片电泳是唯一一种能够产生最适合大型复杂基因组从头测序和组装的长读段的新技术。与目前的毛细管电泳相比，微芯片系统有望通过增加通量、减少试剂消耗和将测序管道的许多步骤集成到单个平台上，从而大幅降低测序成本。虽然基于毛细管的系统需要大约70分钟来提供大约650个碱基的连续序列，但我们报告了使用独特的聚合物基质/吸附聚合物壁涂层组合的微芯片电泳在6.5分钟内测序多达600个碱基。这意味着测序时间比任何先前发表的芯片测序结果减少了三分之二，具有相当的读取长度和序列质量。我们假设这些芯片上的超高速长读取可以实现，因为组合聚合物系统产生了最近发现的DNA电迁移的“杂交”机制，其中DNA分子在通过完整的聚合物网络复制和破坏网络纠缠之间快速交替，以拖动聚合物通过溶液，类似于我们在单分子DNA成像研究中观察到的dsDNA动力学。最重要的是，这些结果揭示了微芯片电泳提供超快速桑格测序的惊人强大能力，这将转化为增加系统吞吐量和降低成本。
To realize the immense potential of large-scale genomic sequencing after the completion of the second human genome (Venter's), the costs for the complete sequencing of additional genomes must be dramatically reduced. Among the technologies being developed to reduce sequencing costs, microchip electrophoresis is the only new technology ready to produce the long reads most suitable for the de novo sequencing and assembly of large and complex genomes. Compared with the current paradigm of capillary electrophoresis, microchip systems promise to reduce sequencing costs dramatically by increasing throughput, reducing reagent consumption, and integrating the many steps of the sequencing pipeline onto a single platform. Although capillary-based systems require approximate to 70 min to deliver approximate to 650 bases of contiguous sequence, we report sequencing up to 600 bases in just 6.5 min by microchip electrophoresis with a unique polymer matrix/adsorbed polymer wall coating combination. This represents a two-thirds reduction in sequencing time over any previously published chip sequencing result, with comparable read length and sequence quality. We hypothesize that these ultrafast long reads on chips can be achieved because the combined polymer system engenders a recently discovered "hybrid" mechanism of DNA electromigration, in which DNA molecules alternate rapidly between reptating through the intact polymer network and disrupting network entanglements to drag polymers through the solution, similar to dsDNA dynamics we observe in single-molecule DNA imaging studies. Most importantly, these results reveal the surprisingly powerful ability of microchip electrophoresis to provide ultrafast Sanger sequencing, which will translate to increased system throughput and reduced costs.