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)...
| Main Authors: | , , , , , |
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| Format: | Journal article |
| Language: | English |
| Published: |
2004
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| _version_ | 1826306016157892608 |
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| author | Linnell, J Mott, R Field, S Kwiatkowski, D Ragoussis, J Udalova, I |
| author_facet | Linnell, J Mott, R Field, S Kwiatkowski, D Ragoussis, J Udalova, I |
| author_sort | Linnell, J |
| collection | OXFORD |
| description | 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. |
| first_indexed | 2024-03-07T06:41:32Z |
| format | Journal article |
| id | oxford-uuid:f9723e4f-882a-41b6-9834-7595d465f9a3 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T06:41:32Z |
| publishDate | 2004 |
| record_format | dspace |
| spelling | oxford-uuid:f9723e4f-882a-41b6-9834-7595d465f9a32022-03-27T12:58:01ZQuantitative high-throughput analysis of transcription factor binding specificities.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:f9723e4f-882a-41b6-9834-7595d465f9a3EnglishSymplectic Elements at Oxford2004Linnell, JMott, RField, SKwiatkowski, DRagoussis, JUdalova, IWe 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. |
| spellingShingle | Linnell, J Mott, R Field, S Kwiatkowski, D Ragoussis, J Udalova, I Quantitative high-throughput analysis of transcription factor binding specificities. |
| title | Quantitative high-throughput analysis of transcription factor binding specificities. |
| title_full | Quantitative high-throughput analysis of transcription factor binding specificities. |
| title_fullStr | Quantitative high-throughput analysis of transcription factor binding specificities. |
| title_full_unstemmed | Quantitative high-throughput analysis of transcription factor binding specificities. |
| title_short | Quantitative high-throughput analysis of transcription factor binding specificities. |
| title_sort | quantitative high throughput analysis of transcription factor binding specificities |
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