Web Release Date: November 3,
Ultrasensitive PCR and Real-Time Detection from Human Genomic Samples Using a Bidirectional Flow Microreactor
and
Institute for Analytical Sciences, Bunsen-Kirchhoff Strasse 11, D-44139 Dortmund, Germany, National Institute of Standards and Technology, Electronics and Engineering Laboratory, 100 Bureau Drive, MS 8120, Building 225, Gaithersburg, Maryland 20899, and The Manchester Interdisciplinary Biocentre, University of Manchester, 131, Princess Street, Manchester. M1 7ND, U.K.
Received for review August 6, 2007. Accepted September 28, 2007.
Abstract:
In this paper we present a reliable bidirectional flow DNA amplification microreactor for processing real-world genomic samples. This system shares the low-power thermal responsiveness of a continuous flow reactor with the low surface area to volume ratio character of stationary reactors for reducing surface inhibitory effects. Silanization with dimethyldichlorosilane in combination with dynamic surface passivation was used to enhance PCR compatibility and enable efficient amplification. For real-time fragment amplification monitoring we have implemented an epimodal fluorescent detection capability. The passivated bidirectional flow system was ultrasensitive, achieving an RNase P gene detection limit of 24 human genome copies with a reaction efficiency of 77%. This starts to rival the performance of a conventional real-time PCR instrument with a reaction efficiency of 93% and revitalizes flow-through PCR as a viable component of lab on a chip DNA analysis formats.
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