Spinning Disk Platform for Microfluidic Digital Polymerase Chain Reaction

University of Utah, Rm 5R441, 1795 E South Campus Dr., Salt Lake City, Utah 84112, USA.
Analytical Chemistry (Impact Factor: 5.83). 02/2010; 82(4):1546-50. DOI: 10.1021/ac902398c
Source: PubMed

ABSTRACT An inexpensive plastic disk disposable was designed for digital polymerase chain reaction (PCR) applications with a microfluidic architecture that passively compartmentalizes a sample into 1000 nanoliter-sized wells by centrifugation. Well volumes of 33 nL were attained with a 16% volume coefficient of variation (CV). A rapid air thermocycler with aggregate real-time fluorescence detection was used, achieving PCR cycle times of 33 s and 94% PCR efficiency, with a melting curve to validate product specificity. A CCD camera acquired a fluorescent image of the disk following PCR, and the well intensity frequency distribution and Poisson distribution statistics were used to count the positive wells on the disk to determine the number of template molecules amplified. A 300 bp plasmid DNA product was amplified within the disk and analyzed in 50 min with 58-1000 wells containing plasmid template. Target concentrations measured by the spinning disk platform were 3 times less than that predicted by absorbance measurements. The spinning disk platform reduces disposable cost, instrument complexity, and thermocycling time compared to other current digital PCR platforms.

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    • "We used capillary PCR and a PCR melting approach for preliminary characterization of extraction output and establishment of RT-PCR protocols. The RT-PCR disks were loaded first by pipetting 10 µL each of PCR mix and mineral oil (M5904, Sigma-Aldrich Corporation), dyed with Oil Red O (Matheson Coleman & Bell, Gardena, CA), into the individual loading reservoirs of the disk and the disk was then spun at 4000 rpm for 5 min to move the fluid from the middle to the outside of the disk [4]. Disk images before and after the PCR runs were taken and samples were centrifuged into collecting vials and subjected to melting or gel electrophoresis as required. "
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