Quantitative high-throughput analysis of transcription factor binding specificities. Export

@article{citeulike:2192710, abstract = {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.}, address = {Wellcome Trust Centre for Human Genetics, University of Oxford, 7 Roosevelt Drive, Oxford OX3 7BN, UK.}, author = {Linnell, J. and Mott, R. and Field, S. and Kwiatkowski, D. P. and Ragoussis, J. and Udalova, I. A.}, citeulike-article-id = {2192710}, citeulike-linkout-0 = {http://dx.doi.org/10.1093/nar/gnh042}, citeulike-linkout-1 = {http://nar.oxfordjournals.org/cgi/content/abstract/32/4/e44}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/14990752}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=14990752}, doi = {10.1093/nar/gnh042}, issn = {1362-4962}, journal = {Nucleic acids research}, keywords = {pbm}, number = {4}, posted-at = {2008-01-04 00:11:23}, priority = {0}, title = {Quantitative high-throughput analysis of transcription factor binding specificities.}, url = {http://dx.doi.org/10.1093/nar/gnh042}, volume = {32}, year = {2004} }

TY - JOUR ID - citeulike:2192710 L3 - citeulike-article-id:2192710 N2 - 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. IS - 4 JF - Nucleic acids research SN - 1362-4962 AD - Wellcome Trust Centre for Human Genetics, University of Oxford, 7 Roosevelt Drive, Oxford OX3 7BN, UK. TI - Quantitative high-throughput analysis of transcription factor binding specificities. VL - 32 KW - pbm AU - Linnell, J AU - Mott, R AU - Field, S AU - Kwiatkowski, DP AU - Ragoussis, J AU - Udalova, IA PY - 2004/// UR - http://dx.doi.org/10.1093/nar/gnh042 ER -