Continuous-Flow Polymerase Chain Reaction of Single-Copy DNA in Microfluidic Microdroplets
Continuous-Flow Polymerase Chain Reaction of Single-Copy DNA in Microfluidic Microdroplets
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DOI：
10.1021/ac802038c
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发表时间：
2009-01-01
影响因子：
7.4
通讯作者：
Hollfelder, Florian
中科院分区：
文献类型：
--
作者：
Schaerli, Yolanda;Wootton, Robert C.;Hollfelder, Florian
我们提出了一种高通量的微流控装置，用于在纳升体积的油包水液滴中进行连续流动聚合酶链反应（PCR）。该装置的圆形设计允许液滴通过交替的温度区，并在仅17分钟内完成34个PCR循环，避免了整个装置的温度循环。所应用的双温PCR方案的温度可以根据模板和引物的要求进行调整。利用罗丹明B的温度依赖性荧光寿命，用液滴内部的荧光寿命成像（FLIM）确定这些温度。证明了从四种不同起始浓度成功扩增85个碱基对长的模板。通过凝胶电泳、测序和实时PCR对产物进行的分析表明，扩增是特异性的，并且高达5 × 10（6）倍的扩增因子与台式PCR仪中获得的扩增因子相当。高效率允许从每个液滴的单个DNA分子扩增。该设备有望方便地与其他微流体设备集成，并为芯片实验室工具包添加了一个关键的缺失组件。
We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 x 10(6)-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.