Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors.
Identifieur interne : 000675 ( Ncbi/Merge ); précédent : 000674; suivant : 000676Universal 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
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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><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">19265799</PMID><DateCompleted><Year>2009</Year><Month>06</Month><Day>12</Day></DateCompleted><DateRevised><Year>2018</Year><Month>11</Month><Day>13</Day></DateRevised><Article PubModel="Print"><Journal><ISSN IssnType="Electronic">1750-2799</ISSN><JournalIssue CitedMedium="Internet"><Volume>4</Volume><Issue>3</Issue><PubDate><Year>2009</Year></PubDate></JournalIssue><Title>Nature protocols</Title><ISOAbbreviation>Nat Protoc</ISOAbbreviation></Journal><ArticleTitle>Universal protein-binding microarrays for the comprehensive characterization of the DNA-binding specificities of transcription factors.</ArticleTitle><Pagination><MedlinePgn>393-411</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1038/nprot.2008.195</ELocationID><Abstract><AbstractText>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. 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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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