Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts.

Détails

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Etat: Public
Version: de l'auteur
ID Serval
serval:BIB_B12289BE4F94
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Proof of concept for microarray-based detection of DNA-binding oncogenes in cell extracts.
Périodique
Nucleic Acids Research
Auteur(s)
Egener T., Roulet E., Zehnder M., Bucher P., Mermod N.
ISSN
1362-4962[electronic], 0305-1048[linking]
Statut éditorial
Publié
Date de publication
2005
Volume
33
Numéro
8
Pages
e79
Langue
anglais
Notes
Publication types: Evaluation Studies ; Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: epublish
Résumé
The function of DNA-binding proteins is controlled not just by their abundance, but mainly at the level of their activity in terms of their interactions with DNA and protein targets. Moreover, the affinity of such transcription factors to their target sequences is often controlled by co-factors and/or modifications that are not easily assessed from biological samples. Here, we describe a scalable method for monitoring protein-DNA interactions on a microarray surface. This approach was designed to determine the DNA-binding activity of proteins in crude cell extracts, complementing conventional expression profiling arrays. Enzymatic labeling of DNA enables direct normalization of the protein binding to the microarray, allowing the estimation of relative binding affinities. Using DNA sequences covering a range of affinities, we show that the new microarray-based method yields binding strength estimates similar to low-throughput gel mobility-shift assays. The microarray is also of high sensitivity, as it allows the detection of a rare DNA-binding protein from breast cancer cells, the human tumor suppressor AP-2. This approach thus mediates precise and robust assessment of the activity of DNA-binding proteins and takes present DNA-binding assays to a high throughput level.
Mots-clé
Base Sequence, Binding Sites, Breast Neoplasms/metabolism, Cell Extracts, Cell Line, Tumor, DNA/chemistry, DNA/metabolism, DNA Probes, DNA-Binding Proteins/analysis, DNA-Binding Proteins/metabolism, Electrophoretic Mobility Shift Assay, Female, Humans, Oligonucleotide Array Sequence Analysis/methods, Oncogene Proteins/analysis, Transcription Factor AP-2, Transcription Factors/analysis, Transcription Factors/metabolism
Pubmed
Web of science
Open Access
Oui
Création de la notice
24/01/2008 10:41
Dernière modification de la notice
20/08/2019 15:20
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