Quantitative high-throughput analysis of transcription factor binding specificities.
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.
Wellcome Trust Centre for Human Genetics, University of Oxford, 7 Roosevelt Drive, Oxford OX3 7BN, UK.
Jane
J
Linnell
Richard
R
Mott
Simon
S
Field
Dominic P
DP
Kwiatkowski
Jiannis
J
Ragoussis
Irina A
IA
Udalova
D000180:Acrylic Resins; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004952:Esters; D005455:Fluorescent Antibody Technique; D005898:Glass; D051863:Host Cell Factor C1; D006801:Humans; D016328:NF-kappa B / Q000378:metabolism; D050811:Octamer Transcription Factor-1; D020411:Oligonucleotide Array Sequence Analysis; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011485:Protein Binding; D015203:Reproducibility of Results; D020218:Response Elements / Q000235:genetics*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000099166:Polyacrylamides
D000180:Acrylic Resins; D004247:DNA; D004268:DNA-Binding Proteins; D004952:Esters; D051863:Host Cell Factor C1; D016328:NF-kappa B; D050811:Octamer Transcription Factor-1; D014157:Transcription Factors; D000099166:Polyacrylamides; C497692:HCFC1 protein, human; C494165:POU2F1 protein, human