Concentration of DNA in a flowing stream for high-sensitivity capillary electrophoresis.
Concentration of DNA in a flowing stream for high-sensitivity capillary electrophoresis.
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DOI：
10.1021/ac034209h
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发表时间：
2003-07
影响因子：
7.4
通讯作者：
Sang-Ryoul Park;H. Swerdlow
中科院分区：
文献类型：
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作者：
Sang-Ryoul Park;H. Swerdlow
描述了一种新型的样品预处理装置，并证明了其在随后的毛细管电泳的流动流中的粗DNA样品的浓缩和纯化的应用。该器械由两个间隙连接组成，每个间隙连接均覆盖有导电膜材料，并建立在PEEK管制成的流动通道上。在结点之间施加电场时，带负电荷的DNA片段可以抵抗流体动力学流动流并被捕获在结点之间。通过简单地推动样品溶液通过装置，以微升体积溶解的DNA片段被捕获在纳升大小的条带中。根据它们的电泳迁移率，粗样品中的其他干扰物质可以通过洗涤从捕获的DNA片段中去除。膜对小离子的选择性渗透性允许有效的过滤。浓缩和纯化的DNA片段通过简单地关闭或反转电场来释放。回收率高达95%。使用荧光染料-引物循环测序反应的粗产物评价装置的性能。与这些粗反应产物相比，当在常规毛细管电泳仪器上运行时，在捕获装置中纯化并随后收集的样品显示出显著增强的信号和分辨率。此外，该装置可以在线连接到毛细管系统，用于直接注射。该器件具有很大的潜力，使芯片实验室系统能够与真实世界的样品一起使用。
A novel sample pretreatment device is described, and its application to the concentration and purification of crude DNA samples in a flowing stream for subsequent capillary electrophoresis is demonstrated. The device consists of two gap junctions, each covered with a conductive membrane material and built upon a flow channel made of PEEK tubing. Upon the application of an electric field between the junctions, the negatively charged DNA fragments can resist the hydrodynamic flow stream and are trapped between the junctions. DNA fragments dissolved in microliter volumes are captured in a nanoliter-sized band by simply pushing the sample solution through the device. Depending on their electrophoretic mobility, other interfering materials in a crude sample can be removed from the trapped DNA fragments by washing. The selective permeability of the membrane to small ions allows efficient desalting. The concentrated and purified DNA fragments are released by simply turning off or reversing the electric field. Recovery is up to 95%. Performance of the device was evaluated using crude products of fluorescent dye-primer cycle-sequencing reactions. Compared to these crude reaction products, samples purified in the capture device and subsequently collected showed dramatically enhanced signal and resolution when run on a conventional capillary-electrophoresis instrument. Furthermore, the device could be connected in-line to a capillary system for direct injection. The device has great potential for enabling lab-on-a-chip systems to be used with real-world samples.