Quantitative high-throughput analysis of transcription factor binding specificities.
Nucleic Acids Res
; 32(4): e44, 2004 Feb 27.
Article
en En
| MEDLINE
| ID: mdl-14990752
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Factores de Transcripción
/
ADN
/
Elementos de Respuesta
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2004
Tipo del documento:
Article
País de afiliación:
Reino Unido
Pais de publicación:
ENGLAND
/
ESCOCIA
/
GB
/
GREAT BRITAIN
/
INGLATERRA
/
REINO UNIDO
/
SCOTLAND
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UK
/
UNITED KINGDOM