High-throughput quantitative polymerase chain reaction in picoliter droplets.
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/microL). 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.
Kiss
Margaret Macris
MM
Raindance Technologies, 44 Hartwell Avenue, Lexington, Massachusetts 02421, USA.
Ortoleva-Donnelly
Lori
L
Beer
N Reginald
NR
Warner
Jason
J
Bailey
Christopher G
CG
Colston
Bill W
BW
Rothberg
Jonathon M
JM
Link
Darren R
DR
Leamon
John H
JH
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis*; D004867:Equipment Design; D005456:Fluorescent Dyes; D016679:Genome, Viral; D046210:Microfluidic Analytical Techniques / Q000191:economics / Q000295:instrumentation* / Q000379:methods; D016133:Polymerase Chain Reaction / Q000191:economics / Q000295:instrumentation / Q000379:methods*; D018401:Sample Size; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral; D005456:Fluorescent Dyes