Integrated bare narrow capillary-hydrodynamic chromatographic system for free-solution DNA separation at the single-molecule level.
Integrated bare narrow capillary-hydrodynamic chromatographic system for free-solution DNA separation at the single-molecule level.
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DOI：
10.1002/anie.201300208
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发表时间：
2013-05-17
影响因子：
16.6
通讯作者：
Liu, Shaorong
中科院分区：
文献类型：
--
作者：
Zhu, Zaifang;Chen, Huang;Wang, Wei;Morgan, Aaron;Gu, Congying;He, Chiyang;Lu, Joann J.;Liu, Shaorong
自由溶液中单分子水平的DNA。DNA分离是分子生物学研究中的一项常见任务。传统上，DNA分子是用平板凝胶电泳分离的，包括脉冲场凝胶电泳（PFGE）为了提高分辨率、减少运行时间和提高通量，毛细管凝胶电泳（CGE）和后来的毛细管阵列电泳（CAE）得到了发展，并取得了较高的分辨率然而，CGE和CAE都需要粘性聚合物筛分基质，这很难处理，特别是当使用狭窄的毛细血管时。研究人员已经进行了在自由溶液中分离DNA的实验，但是DNA在这些介质中不容易分离，因为所有的DNA分子都具有相似的质量电荷比（m/z），因此电泳迁移率也相似。1992年，Noolandi[4]提出了一种解决这一问题的方法，将一个单分散的实体附着在每个DNA片段上，产生不同的待分离DNA的m/z值。这个想法在20世纪90年代末得到了实验验证，并被称为末端标记自由溶液电泳（ELFSE）。这种方法已被证明可以有效地分离短于几百个碱基对的DNA片段其他用于DNA分离的无凝胶方法包括径向迁移、[7]液相色谱、[8]熵捕获、[9]和DNA棱镜这些方法克服了粘性凝胶带来的问题，为DNA的解析提供了有希望的替代方法，但与凝胶电泳相比，它们的分辨率没有竞争力。最近，我们开发了一种新技术，称为bcb - hdc，[11]，用于自由溶液DNA分离。当DNA分子在压力驱动的条件下在狭窄的毛细管内运输时，DNA分子以颗粒的形式移动较大的DNA片段具有更大的有效直径，不能像较小的片段那样靠近毛细血管壁（慢速移动区域），因此，它们移动得更快。基于这一原理，已经用与凝胶电泳相当的分辨率分离出了尺寸范围很广的DNA片段样品最小的废物产生和较低的操作成本使bac - hdc成为凝胶基技术的有吸引力的替代品，特别是用于分离大DNA片段的PFGE。在此，我们将高压电渗透泵（EOP）和微加工芯片注入器与bac - hdc集成在一起；集成系统使我们能够可靠地以低皮升（pL）体积注射样品，以数百pLmin−1的流速或更低的可重复性洗脱分析物，并在单分子水平上在自由溶液中快速解析大范围的DNA片段。
DNA at the single-molecule level in free solution. DNA separation is a common task in molecular biological research. Traditionally, DNA molecules are separated using slab-gel electrophoresis,[1] including pulsed field gel electrophoresis (PFGE).[2] To improve resolution, reduce running time, and increase throughput, capillary gel electrophoresis (CGE) and later capillary array electrophoresis (CAE) have been developed, and high resolutions have been achieved.[3] However, both CGE and CAE require viscous polymer sieving matrices, which can be difficult to work with, especially when narrow capillaries are employed. Researchers have experimented with separating DNA in free solutions, but DNA cannot be easily resolved in these media because all DNA molecules have similar mass-tocharge ratios (m/z), and, as a result, similar electrophoretic mobilities. In 1992, Noolandi [4] proposed an approach to solve this problem by attaching a monodisperse entity to each DNA fragment, generating varying m/z values for the DNA to be separated. The idea was experimentally validated in the late 1990s [5] and termed end-labeled free-solution electrophoresis (ELFSE). This method has proven to be effective for separating DNA fragments shorter than a few hundreds of base pairs.[6] Other gel-free approaches for DNA separation include radial migration,[7] liquid chromatography,[8] entropic trapping,[9] and DNA prism.[10] These approaches overcome the problems brought by viscous gels and offer promising alternatives for resolving DNA, but their resolutions are not competitive compared to that of gel electrophoresis. Recently, we have developed a new technique, called BaNC-HDC,[11] for free-solution DNA separations. When DNA molecules are transported inside a narrow capillary under pressure-driven conditions, the DNA molecules move as particles.[12] Larger DNA fragments have greater effective diameters and cannot go as close to the capillary wall (the slow-moving region) as smaller fragments can and, therefore, they move faster. On the basis of this principle, samples of DNA fragments with a wide size range have been separated with resolutions comparable to gel electrophoresis.[11] The minimal waste generation and low operation costs make BaNC-HDC an attractive alternative to gel-based techniques, particularly to PFGE for separating large DNA fragments. Herein, we integrate a high-pressure electroosmotic pump (EOP) and a microfabricated chip-injector with BaNC-HDC; the integrated system enables us to inject samples at low-picoliter (pL) volumes reliably, elute analytes at hundreds of pLmin− 1 flowrates or lower reproducibly, and resolve a wide size range of DNA fragments rapidly in free solution at the single-molecule level.
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影响因子：
4.1
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