Continuous-Flow Polymerase Chain Reaction of Single-Copy DNA in Microfluidic Microdroplets
| Autor: | Robert C. R. Wootton, Andrew J. deMello, Yolanda Schaerli, Paul M. W. French, Christopher Dunsby, Florian Hollfelder, Tom Robinson, Viktor Stein, Mark Andrew Aquilla Neil, Chris Abell |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Electrophoresis
Fluorescence-lifetime imaging microscopy Microfluidics Analytical chemistry Recombinase Polymerase Amplification 02 engineering and technology Temperature cycling Polymerase Chain Reaction 01 natural sciences Analytical Chemistry law.invention Rhodamine chemistry.chemical_compound Polymerase chain reaction optimization law Polymethyl Methacrylate Polymerase chain reaction Fluorescent Dyes Rhodamines 010401 analytical chemistry Temperature Water DNA Microfluidic Analytical Techniques 021001 nanoscience & nanotechnology 0104 chemical sciences chemistry 0210 nano-technology Oils |
| Zdroj: | Analytical Chemistry |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/ac802038c |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|