Continuous-flow polymerase chain reaction of single-copy DNA in microfluidic microdroplets.

Détails

ID Serval
serval:BIB_509F32E759B0
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Continuous-flow polymerase chain reaction of single-copy DNA in microfluidic microdroplets.
Périodique
Analytical Chemistry
Auteur(s)
Schaerli Y., Wootton R.C., Robinson T., Stein V., Dunsby C., Neil M.A., French P.M., Demello A.J., Abell C., Hollfelder F.
ISSN
1520-6882 (Electronic)
ISSN-L
0003-2700
Statut éditorial
Publié
Date de publication
2009
Peer-reviewed
Oui
Volume
81
Numéro
1
Pages
302-306
Langue
anglais
Résumé
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.

Mots-clé
DNA/chemistry, DNA/genetics, Electrophoresis/methods, Fluorescent Dyes/chemistry, Microfluidic Analytical Techniques/instrumentation, Microfluidic Analytical Techniques/methods, Oils/chemistry, Polymerase Chain Reaction/instrumentation, Polymerase Chain Reaction/methods, Polymethyl Methacrylate/chemistry, Rhodamines/chemistry, Temperature, Water/chemistry
Pubmed
Web of science
Création de la notice
02/02/2017 9:15
Dernière modification de la notice
03/03/2018 17:09
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