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Published online 27 February 2004

Nucleic Acids Research, 2004, Vol. 32, No. 4 e44
© 2004 Oxford University Press

Quantitative high-throughput analysis of transcription factor binding specificities

Jane Linnell, Richard Mott, Simon Field, Dominic P. Kwiatkowski, Jiannis Ragoussis and Irina A. Udalova*

Wellcome Trust Centre for Human Genetics, University of Oxford, 7 Roosevelt Drive, Oxford OX3 7BN, UK

*To whom correspondence should be addressed. Tel: +44 1865 287671; Fax: +44 1865 287533; Email: iudalova{at}molbiol.ox.ac.uk
Correspondence may also be addressed to Jiannis Ragoussis. Email: ioannis.ragoussis{at}well.ox.ac.uk

We present a general high-throughput approach to accurately quantify DNA–protein interactions, which can facilitate the identification of functional genetic polymorphisms. The method tested here on two structurally distinct transcription factors (TFs), NF-{kappa}B and OCT-1, comprises three steps: (i) optimized selection of DNA variants to be tested experimentally, which we show is superior to selecting variants at random; (ii) a quantitative protein–DNA binding assay using microarray and surface plasmon resonance technologies; (iii) prediction of binding affinity for all DNA variants in the consensus space using a statistical model based on principal coordinates analysis. For the protein–DNA binding assay, we identified a polyacrylamide/ester glass activation chemistry which formed exclusive covalent bonds with 5'-amino-modified DNA duplexes and hindered non-specific electrostatic attachment of DNA. Full accessibility of the DNA duplexes attached to polyacrylamide-modified slides was confirmed by the high degree of data correlation with the electromobility shift assay (correlation coefficient 93%). This approach offers the potential for high-throughput determination of TF binding profiles and predicting the effects of single nucleotide polymorphisms on TF binding affinity. New DNA binding data for OCT-1 are presented.


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