Performance of nanoliter-sized droplet-based microfluidic PCR |
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Authors: | Fang Wang Mark A Burns |
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Institution: | (1) Department of Chemical Engineering, University of Michigan, Ann Arbor, MI 48109, USA;(2) Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA; |
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Abstract: | A microfluidic device was used to characterize PCR in aqueous-in-oil droplets for potential point-of-care applications. Droplets
with a volume range of 5–250 nL can be formed on-chip reproducibly, and PCR in the droplets shows amplification efficiencies
comparable to benchtop reactions with no evaporation loss. A higher polymerase concentration is required in the reaction droplet
while the optimal Magnesium ion concentration is the same for both on-chip and benchtop systems. The optimal hold time is
9 s and 30 s for denaturation and annealing/extension in thermal cycling, respectively. With the optimized cycling parameters,
the total reaction time is reduced to half of that required for benchtop PCR. For the droplets containing the same quantity
of template DNA, the PCR yield is approximately the same with either fixed droplet size or fixed template DNA concentration.
The droplet-based PCR can be monitored in real time with FRET probes, and provide amplification with a cycle threshold of
~10 cycles earlier than the benchtop instruments. |
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Keywords: | |
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