On-chip, real-time, single-copy polymerase chain reaction in picoliter droplets.
The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 106 smaller than commercial real-time PCR instruments. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil-phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermally cycled through the PCR protocol without droplet motion. With this system, a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of approximately 18, 20 cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.
Beer
N Reginald
NR
Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94551, USA.
Hindson
Benjamin J
BJ
Wheeler
Elizabeth K
EK
Hall
Sara B
SB
Rose
Klint A
KA
Kennedy
Ian M
IM
Colston
Bill W
BW
D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D013997:Time Factors