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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 Res

Publication Date





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