In vitro analysis of DNA-protein interactions by proximity ligation
Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro metho...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2007-02, Vol.104 (9), p.3067-3072 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Gustafsdottir, Sigrun M Schlingemann, Joerg Rada-Iglesias, Alvaro Schallmeiner, Edith Kamali-Moghaddam, Masood Wadelius, Claes Landegren, Ulf |
| description | Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1-10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites. |
| doi_str_mv | 10.1073/pnas.0611229104 |
| format | Article |
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Information about such elements is currently in a state of rapid growth, but improved methods are required to characterize the sequence specificity of DNA-binding proteins. We have established an in vitro method for specific and sensitive solution-phase analysis of interactions between proteins and nucleic acids in nuclear extracts, based on the proximity ligation assay. The reagent consumption is very low, and the excellent sensitivity of the assay enables analysis of as few as 1-10 cells. We show that our results are highly reproducible, quantitative, and in good agreement with both EMSA and predictions obtained by using a motif finding software. This assay can be a valuable tool to characterize in-depth the sequence specificity of DNA-binding proteins and to evaluate effects of polymorphisms in known transcription factor binding sites.</description><identifier>ISSN: 0027-8424</identifier><identifier>ISSN: 1091-6490</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.0611229104</identifier><identifier>PMID: 17360610</identifier><language>eng</language><publisher>United States: National Academy of Sciences</publisher><subject>Antibodies ; Binding sites ; Biochemistry ; Biological Sciences ; Deoxyribonucleic acid ; DNA ; DNA - metabolism ; DNA binding proteins ; DNA probes ; DNA-Binding Proteins - metabolism ; Electrophoretic Mobility Shift Assay ; Genetic Techniques ; Genomics ; Ligation ; MEDICIN ; MEDICINE ; Models, Molecular ; Molecular Probes - genetics ; Molecular Probes - metabolism ; Molecular Probes/genetics/metabolism ; Oligonucleotide probes ; Oligonucleotides ; Oligonucleotides - genetics ; Oligonucleotides - metabolism ; Oligonucleotides/genetics/metabolism ; Polymerase Chain Reaction ; Proteins ; Transcription factors</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 2007-02, Vol.104 (9), p.3067-3072</ispartof><rights>Copyright 2007 The National Academy of Sciences of the United States of America</rights><rights>Copyright National Academy of Sciences Feb 27, 2007</rights><rights>2007 by The National Academy of Sciences of the USA 2007</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c586t-d284bfb8b9d2a609340f09aad7f963278d634201239cc4a5d8abc32d64b49ec43</citedby><cites>FETCH-LOGICAL-c586t-d284bfb8b9d2a609340f09aad7f963278d634201239cc4a5d8abc32d64b49ec43</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/104/9.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/25426605$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/25426605$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,723,776,780,799,881,27901,27902,53766,53768,57992,58225</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17360610$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-16648$$DView record from Swedish Publication Index$$Hfree_for_read</backlink></links><search><creatorcontrib>Gustafsdottir, Sigrun M</creatorcontrib><creatorcontrib>Schlingemann, Joerg</creatorcontrib><creatorcontrib>Rada-Iglesias, Alvaro</creatorcontrib><creatorcontrib>Schallmeiner, Edith</creatorcontrib><creatorcontrib>Kamali-Moghaddam, Masood</creatorcontrib><creatorcontrib>Wadelius, Claes</creatorcontrib><creatorcontrib>Landegren, Ulf</creatorcontrib><title>In vitro analysis of DNA-protein interactions by proximity ligation</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>Protein-binding DNA sequence elements encode a variety of regulated functions of genomes. 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| subjects | Antibodies Binding sites Biochemistry Biological Sciences Deoxyribonucleic acid DNA DNA - metabolism DNA binding proteins DNA probes DNA-Binding Proteins - metabolism Electrophoretic Mobility Shift Assay Genetic Techniques Genomics Ligation MEDICIN MEDICINE Models, Molecular Molecular Probes - genetics Molecular Probes - metabolism Molecular Probes/genetics/metabolism Oligonucleotide probes Oligonucleotides Oligonucleotides - genetics Oligonucleotides - metabolism Oligonucleotides/genetics/metabolism Polymerase Chain Reaction Proteins Transcription factors |
| title | In vitro analysis of DNA-protein interactions by proximity ligation |
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