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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-kappaB 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.

Original publication




Journal article


Nucleic acids research

Publication Date





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


Humans, Esters, Glass, Acrylic Resins, DNA-Binding Proteins, NF-kappa B, Transcription Factors, DNA, Fluorescent Antibody Technique, Electrophoretic Mobility Shift Assay, Oligonucleotide Array Sequence Analysis, Surface Plasmon Resonance, Sensitivity and Specificity, Reproducibility of Results, Base Sequence, Response Elements, Protein Binding, Substrate Specificity, Polymorphism, Single Nucleotide, Octamer Transcription Factor-1, Host Cell Factor C1