Nanoliter-scale sample preparation methods directly coupled to polymethylmethacrylate-based microchips and gel-filled capillaries for the analysis of oligonucleotides.
Nanoliter-scale sample preparation methods directly coupled to polymethylmethacrylate-based microchips and gel-filled capillaries for the analysis of oligonucleotides.
复制标题
纳升规模的样品制备方法直接与基于聚甲基丙烯酸甲酯的微芯片和凝胶填充毛细管耦合，用于寡核苷酸分析。
DOI：
10.1016/s0021-9673(99)00651-2
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Bruch,RC
中科院分区：
文献类型：
--
作者：
Soper,SA;Ford,SM;Xu,Y;Qi,S;McWhorter,S;Lassiter,S;Patterson,D;Bruch,RC
我们目前正在开发小型化的芯片电泳装置，用塑料制造，用于高速分离寡核苷酸。与这些设备相关的主要优点之一是样品要求小，通常在纳升到亚纳升范围内。不幸的是，大多数标准样品制备方案，特别是寡核苷酸，都是在微升尺度上完成的。我们的工作重点是开发毛细管纳米反应器耦合到微分离平台，如微电泳芯片，用于制备测序阶梯和聚合酶链反应（pcr）。这些纳米反应器由熔融石英毛细管（10-20 cm×20-50 μm I.D.）组成，利用管产生的电渗透流来泵送流体。这些反应器位于快速热循环器中，对dna进行循环测序或PCR扩增。反应器既可以通过用深x射线蚀刻（宽度50 μm，深度50 μm）的聚甲基丙烯酸甲酯（PMMA）微机械加工的毛细管连接器连接到微电泳芯片上，也可以使用零死体积玻璃接头连接到传统的毛细管凝胶管上。对于我们的芯片，它们还包含一个进样器，分离通道（长6厘米，宽30 μm，深50 μm）和双光纤，近红外荧光检测器。测序纳米反应器使用表面固定化模板，通过生物素-链亲和素-生物素连接连接到壁上。测序轨迹可以直接注射到凝胶填充的毛细管中，分离过程的效率降低最小。该纳米反应器还可以配置为通过在毛细管中填充PCR试剂和模板来进行PCR反应。经过热循环后，聚合酶链反应鸡尾酒可以从多个反应器中汇集并装载到平板凝胶或注入毛细管或微芯片设备中进行分离。
We are currently developing miniaturized, chip-based electrophoresis devices fabricated in plastics for the high-speed separation of oligonucleotides. One of the principal advantages associated with these devices is their small sample requirements, typically in the nanoliter to sub-nanoliter range. Unfortunately, most standard sample preparation protocols, especially for oligonucleotides, are done off-chip on a microliter-scale. Our work has focused on the development of capillary nanoreactors coupled to micro-separation platforms, such as micro-electrophoresis chips, for the preparation of sequencing ladders and also polymerase chain reactions (PCRs). These nanoreactors consist of fused-silica capillary tubes (10–20 cm×20–50 μm I.D.) with fluid pumping accomplished using the electroosmotic flow generated by the tubes. These reactors were situated in fast thermal cyclers to perform cycle sequencing or PCR amplification of the DNAs. The reactors could be interfaced to either a micro-electrophoresis chips via capillary connectors micromachined in polymethylmethacrylate (PMMA) using deep X-ray etching (width 50 μm; depth 50 μm) or conventional capillary gel tubes using zero-dead volume glass unions. For our chips, they also contained an injector, separation channel (length 6 cm; width 30 μm; depth 50 μm) and a dual fiber optic, near-infrared fluorescence detector. The sequencing nanoreactor used surface immobilized templates attached to the wall via a biotin–streptavidin–biotin linkage. Sequencing tracks could be directly injected into gel-filled capillary tubes with minimal degradation in the efficiency of the separation process. The nanoreactor could also be configured to perform PCR reactions by filling the capillary tube with the PCR reagents and template. After thermal cycling, the PCR cocktail could be pooled from multiple reactors and loaded onto a slab gel or injected into a capillary tube or microchip device for fractionation.