TaqMan probe array for quantitative detection of DNA targets

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 transferbased microarray platform for real-time quantification of nucleic acid targets that combines advantages of both and reduces their limitations. A set of 30 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, 50 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. INTRODUCTION Microarry-based analyses have been well established and are currently used in a wide range of biological assays (1–5). Extrapolation of their use for infectious diagnostics and detection of biodefense related agents provides an attractive alternative to conventional analytical approaches. Hence the critical need for advanced diagnostic systems in microbiology is to detect rapidly genetic information within the known and unanticipated pathogenic microrganisms associated with the human health (i.e. viruses, bacteria and fungi). With the parallelism offered by DNA microarray technology we are able to pursue and develop an approach to large-scale analyses of such abundance of genetic information in these organisms. The main technical challenge in this field arises from the difficulty in labeling sufficient copies of pathogenic biomarkers. However, when integrated in one portable diagnostic device labelfree detection has great potential for addressing this neckbottle and speeds up practical application of microarray technology. Real-time PCR technologies as label-free methods have been used widely for gene expression, allelic discrimination and pathogen detection in solution (6–10). Their principle is fluorescence resonance energy transfer, where fluorescence is detected as a result of a change in physical distance between a reporter fluorophore and a quencher molecule. Molecular beacons firstly introduced by Tyagi and Kramer (9) and TaqMan probes originated from 50 nuclease cleavage activity (11) and subsequently refined by Lee et al. (12) are the two main probe types in the real-time PCR systems for detecting the accumulation of specific PCR product and discriminating alleles. Their application is usually limited detecting only a few DNA targets, although several efforts have been made to improve the throughput performance of TaqMan probes in *To whom the correspondence should be addressed. Tel/Fax: +86 25 83619983; Email: [email protected] 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 [email protected] Nucleic Acids Research, 2006, Vol. 34, No. 1 e4 doi:10.1093/nar/gnj006