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Nucleic Acids Research 2006 34(1):e4; doi:10.1093/nar/gnj006
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Published online 10 January 2006

© The Author 2006. Published by Oxford University Press. 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, please contact journals.permissions{at}oxfordjournals.org

Methods Online

TaqMan probe array for quantitative detection of DNA targets

Heping Liu1,2, Hong Wang1,4, Zhiyang Shi2, Hua Wang2, Chaoyong Yang4, Spering Silke3, Weihong Tan4 and Zuhong Lu1,*

1State Key Laboratory of Bioelectronics, Southeast University Nanjing 210096, China 2Key Laboratory of Enteric Pathogenic Microbiology (Ministry of Health), Center for Disease Prevention and Control of Jiangsu Province 172 Jiangsu Road, Nanjing 210009, China 3Max-Planck-Institute for Molecular Genetics Ihnestrasse 73, Berlin 14195, Germany 4Center for Research at the Bio/nano Interface, Department of Chemistry and the McKnight Brain Institute, University of Florida Gainesville, FL 32611-7200, USA

*To whom the correspondence should be addressed. Tel/Fax: +86 25 83619983; Email: zhlu{at}seu.edu.cn

Received September 29, 2005. Revised November 7, 2005. Accepted December 13, 2005.

To date real-time quantitative PCR and gene expression microarrays are the methods of choice for quantification of nucleic acids. Herein, we described a unique fluorescence resonance energy transfer-based microarray platform for real-time quantification of nucleic acid targets that combines advantages of both and reduces their limitations. A set of 3' amino-modified TaqMan probes were designed and immobilized on a glass slide composing a regular microarray pattern, and used as probes in the consecutive PCR carried out on the surface. During the extension step of the PCR, 5' nuclease activity of DNA polymerase will cleave quencher dyes of the immobilized probe in the presence of nucleic acids targets. The increase of fluorescence intensities generated by the change in physical distance between reporter fluorophore and quencher moiety of the probes were collected by a confocal scanner. Using this new approach we successfully monitored five different pathogenic genomic DNAs and analyzed the dynamic characteristics of fluorescence intensity changes on the TaqMan probe array. The results indicate that the TaqMan probe array on a planar glass slide monitors DNA targets with excellent specificity as well as high sensitivity. This set-up offers the great advantage of real-time quantitative detection of DNA targets in a parallel array format.


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