In vitro analysis of DNA-protein interactions by proximity ligation

Gustafsdottir, Sigrun M.Uppsala universitet,Institutionen för genetik och patologi (author)

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.

Schlingemann, JörgUppsala universitet,Institutionen för genetik och patologi (author)
Rada-Iglesias, AlvaroUppsala universitet,Institutionen för genetik och patologi (author)
Schallmeiner, EdithUppsala universitet,Institutionen för genetik och patologi(Swepub:uu)edsch087 (author)
Kamali-Moghaddam, MasoodUppsala universitet,Institutionen för genetik och patologi(Swepub:uu)masookam (author)
Wadelius, ClaesUppsala universitet,Institutionen för genetik och patologi(Swepub:uu)claeswad (author)
Landegren, UlfUppsala universitet,Institutionen för genetik och patologi(Swepub:uu)ulfland (author)
Uppsala universitetInstitutionen för genetik och patologi (creator_code:org_t)

In:Proceedings of the National Academy of Sciences of the United States of America: Proceedings of the National Academy of Sciences104:9, s. 3067-30720027-84241091-6490

By the author/editor: Gustafsdottir, S ...; Schlingemann, Jö ...; Rada-Iglesias, A ...; Schallmeiner, Ed ...; Kamali-Moghaddam ...; Wadelius, Claes; show more...; Landegren, Ulf; show less...

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