title: On-chip, real-time, single-copy polymerase chain reaction in picoliter droplets.
abstract: 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.
authors.affiliation: Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94551, USA.
authors.forename: N Reginald
authors.initials: NR
authors.lastname: Beer
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Hindson
authors.forename: Elizabeth K
authors.initials: EK
authors.lastname: Wheeler
authors.forename: Sara B
authors.initials: SB
authors.lastname: Hall
authors.forename: Klint A
authors.initials: KA
authors.lastname: Rose
authors.forename: Ian M
authors.initials: IM
authors.lastname: Kennedy
authors.forename: Bill W
authors.initials: BW
authors.lastname: Colston
mesh_terms: D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D013997:Time Factors