High-throughput quantitative polymerase chain reaction in picoliter droplets.
High-throughput quantitative polymerase chain reaction in picoliter droplets.
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DOI：
10.1021/ac801276c
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发表时间：
2008-12-01
影响因子：
7.4
通讯作者：
Leamon, John H.
中科院分区：
文献类型：
--
作者：
Kiss, Margaret Macris;Ortoleva-Donnelly, Lori;Beer, N. Reginald;Warner, Jason;Bailey, Christopher G.;Colston, Bill W.;Rothberg, Jonathon M.;Link, Darren R.;Leamon, John H.
有限稀释PCR已成为一种越来越有用的技术，用于检测和定量种群中的稀有物种，但检测限和定量的准确性在很大程度上取决于可以分析的反应数量。通过减少反应体积和增加持续合成能力可以实现增加的生产量。我们设计了一种高通量微流控芯片，它可以在连续的油流中将PCR试剂封装在数百万皮升的液滴中。油流引导液滴通过交替的变性和退火区，导致快速（55秒循环）和有效的PCR扩增。在PCR反应混合物中包含荧光探针允许在微流体芯片内的特定位置处的单个液滴内监测扩增过程。我们表明，245 bp腺病毒产物的扩增可以在35分钟内检测和定量，起始模板浓度低至每167个液滴1个模板分子（0.003 pg/μL）。在一系列模板浓度范围内的阳性反应频率与泊松统计预测的频率非常一致，证明了该平台对于有限稀释和数字PCR应用的准确性和灵敏度。
Limiting dilution PCR has become an increasingly useful technique for the detection and quantification of rare species in a population, but the limit of detection and accuracy of quantification are largely determined by the number of reactions that can be analyzed. Increased throughput may be achieved by reducing the reaction volume and increasing processivity. We have designed a high-throughput microfluidic chip that encapsulates PCR reagents in millions of picoliter droplets in a continuous oil flow. The oil stream conducts the droplets through alternating denaturation and annealing zones, resulting in rapid (55 second cycles) and efficient PCR amplification. Inclusion of fluorescent probes in the PCR reaction mix permits the amplification process to be monitored within individual droplets at specific locations within the microfluidic chip. We show that amplification of a 245 bp Adenovirus product can be detected and quantified in 35 minutes at starting template concentrations as low as one template molecule per 167 droplets (0.003 pg/μL). The frequencies of positive reactions over a range of template concentrations agree closely with the frequencies predicted by Poisson statistics, demonstrating both the accuracy and sensitivity of this platform for limiting dilution and digital PCR applications.
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