In vitro analysis of DNA-protein interactions by proximity ligation
2007 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 104, no 9, 3067-3072 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2007. Vol. 104, no 9, 3067-3072 p.
DNA/*metabolism, DNA-Binding Proteins/*metabolism, Electrophoretic Mobility Shift Assay, Genetic Techniques, Models; Molecular, Molecular Probes/genetics/metabolism, Oligonucleotides/genetics/metabolism, Polymerase Chain Reaction
Medical and Health Sciences
IdentifiersURN: urn:nbn:se:uu:diva-16648DOI: 10.1073/pnas.0611229104ISI: 000244661400011PubMedID: 17360610OAI: oai:DiVA.org:uu-16648DiVA: diva2:44419