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Nucleic Acids Research 2005 33(8):e79; doi:10.1093/nar/gni079
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Published online 12 May 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
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Methods Online

Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts

Tanja Egener, Emmanuelle Roulet, Marc Zehnder, Philipp Bucher1 and Nicolas Mermod*

Institute of Biotechnology, University of Lausanne 1015 Lausanne, Switzerland 1Swiss Institute of Bioinformatics and Swiss Institute for Experimental Cancer Research 1066 Epalinges, Switzerland

*To whom correspondence should be addressed at Laboratory of Molecular Biotechnology, Station 6, FSB-ISP-ISIC, EPFL, 1015 Lausanne, Switzerland. Tel: +41 21 693 61 51; Fax: +41 21 693 76 10; Email: Nicolas.Mermod{at}unil.ch

Received February 4, 2005. Revised April 12, 2005. Accepted April 27, 2005.

The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein–DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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