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Nucleic Acids Research 2005 33(2):e11; doi:10.1093/nar/gnh184
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Published online 20 January 2005

© 2005, the authors Nucleic Acids Research, Vol. 33 No. 2 © Oxford University Press 2005; all rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use permissions, please contact journals.permissions{at}oupjournals.org.

Methods Online

DNA analysis with multiplex microarray-enhanced PCR

A. Pemov, H. Modi, D. P. Chandler and S. Bavykin*

Biodetection Technologies Section, Energy Systems Division, Argonne National Laboratory 9700 South Cass Avenue, Argonne, IL 60439, USA

*To whom correspondence should be addressed. Tel: +1 630 252 3980; Fax: +1 630 252 9155; Email: sbavykin{at}anl.gov

Received September 15, 2004. Revised November 29, 2004. Accepted November 29, 2004.

We have developed a highly sensitive method for DNA analysis on 3D gel element microarrays, a technique we call multiplex microarray-enhanced PCR (MME-PCR). Two amplification strategies are carried out simultaneously in the reaction chamber: on or within gel elements, and in bulk solution over the gel element array. MME-PCR is initiated by multiple complex primers containing gene-specific, forward and reverse, sequences appended to the 3' end of a universal amplification primer. The complex primer pair is covalently tethered through its 5' end to the polyacryl- amide backbone. In the bulk solution above the gel element array, a single pair of unattached universal primers simultaneously directs pseudo-monoplex PCR of all targets according to normal solution-phase PCR. The presence of a single universal PCR primer pair in solution accelerates amplification within gel elements and eliminates the problem of primer interference that is common to conventional multiplex PCR. We show 106-fold amplification of targeted DNA after 50 cycles with average amplification efficiency 1.34 per cycle, and demonstrate specific on-chip amplification of six genes in Bacillus subtilis. All six genes were detected at 4.5 pg of bacterial genomic DNA (equivalent to 103 genomes) in 60 independent amplification reactions performed simultaneously in single reaction chamber.


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