title: Continuous-flow polymerase chain reaction of single-copy DNA in microfluidic microdroplets.
abstract: We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 x 10(6)-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.
authors.affiliation: Department of Biochemistry and Department of Chemistry, University of Cambridge, Cambridge, UK.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Schaerli
authors.forename: Robert C
authors.initials: RC
authors.lastname: Wootton
authors.forename: Tom
authors.initials: T
authors.lastname: Robinson
authors.forename: Viktor
authors.initials: V
authors.lastname: Stein
authors.forename: Christopher
authors.initials: C
authors.lastname: Dunsby
authors.forename: Mark A A
authors.initials: MA
authors.lastname: Neil
authors.forename: Paul M W
authors.initials: PM
authors.lastname: French
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Demello
authors.forename: Chris
authors.initials: C
authors.lastname: Abell
authors.forename: Florian
authors.initials: F
authors.lastname: Hollfelder
mesh_terms: D004247:DNA / Q000737:chemistry* / Q000235:genetics; D004586:Electrophoresis / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation / Q000379:methods*; D009821:Oils / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D019904:Polymethyl Methacrylate / Q000737:chemistry; D012235:Rhodamines / Q000737:chemistry; D013696:Temperature; D014867:Water / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D009821:Oils; D012235:Rhodamines; D014867:Water; D004247:DNA; D019904:Polymethyl Methacrylate; C029773:rhodamine B