Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors.
Identifieur interne : 002760 ( Main/Exploration ); précédent : 002759; suivant : 002761Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors.
Auteurs : Michael F. Berger [États-Unis] ; Martha L. BulykSource :
- Nature protocols [ 1750-2799 ] ; 2009.
Descripteurs français
- KwdFr :
- MESH :
English descriptors
- KwdEn :
- MESH :
- chemical , chemistry : DNA.
- chemical , metabolism : DNA-Binding Proteins, Transcription Factors.
- methods : Protein Array Analysis.
- Binding Sites, Image Processing, Computer-Assisted, Sequence Analysis, DNA.
Abstract
Protein-binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA-binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding-site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF-binding specificities at high resolution using such 'all 10-mer' universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day.
DOI: 10.1038/nprot.2008.195
PubMed: 19265799
Affiliations:
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Bulyk</name></author></analytic><series><title level="j">Nature protocols</title><idno type="eISSN">1750-2799</idno><imprint><date when="2009" type="published">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Binding Sites</term><term>DNA (chemistry)</term><term>DNA-Binding Proteins (metabolism)</term><term>Image Processing, Computer-Assisted</term><term>Protein Array Analysis (methods)</term><term>Sequence Analysis, DNA</term><term>Transcription Factors (metabolism)</term></keywords><keywords scheme="KwdFr" xml:lang="fr"><term>ADN ()</term><term>Analyse de séquence d'ADN</term><term>Analyse par réseau de protéines ()</term><term>Facteurs de transcription (métabolisme)</term><term>Protéines de liaison à l'ADN (métabolisme)</term><term>Sites de fixation</term><term>Traitement d'image par ordinateur</term></keywords><keywords scheme="MESH" type="chemical" qualifier="chemistry" xml:lang="en"><term>DNA</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>DNA-Binding Proteins</term><term>Transcription Factors</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>Protein Array Analysis</term></keywords><keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Facteurs de transcription</term><term>Protéines de liaison à l'ADN</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Binding Sites</term><term>Image Processing, Computer-Assisted</term><term>Sequence Analysis, DNA</term></keywords><keywords scheme="MESH" xml:lang="fr"><term>ADN</term><term>Analyse de séquence d'ADN</term><term>Analyse par réseau de protéines</term><term>Sites de fixation</term><term>Traitement d'image par ordinateur</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Protein-binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA-binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding-site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF-binding specificities at high resolution using such 'all 10-mer' universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country></list><tree><noCountry><name sortKey="Bulyk, Martha L" sort="Bulyk, Martha L" uniqKey="Bulyk M" first="Martha L" last="Bulyk">Martha L. Bulyk</name></noCountry><country name="États-Unis"><noRegion><name sortKey="Berger, Michael F" sort="Berger, Michael F" uniqKey="Berger M" first="Michael F" last="Berger">Michael F. Berger</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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