Journal
ANALYTICAL CHEMISTRY
Volume 80, Issue 23, Pages 8975-8981Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac801276c
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Funding
- NIH [NCI 1R21 CA12569301]
- U.S. Department of Energy by Lawrence Livermore National Laboratory [DE-AC52-07NA27344]
- Laboratory Directed Research and Development Program at LLNL [06-ERD-064]
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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-s 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 min at starting template concentrations as low as 1 template molecule/167 droplets (0.003 pg/mu L). 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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