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Visualising individual sequence-specific protein-DNA interactions in situ
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab. (Landegren)
Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis and Human-Computer Interaction. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Visual Information and Interaction. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
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2012 (English)In: New Biotechnology, ISSN 1871-6784, E-ISSN 1876-4347, Vol. 29, no 5, 589-598 p.Article in journal (Refereed) Published
Abstract [en]

Gene expression-a key feature for modulating cell fate-is regulated in part by histone modifications, which modulate accessibility of the chromatin to transcription factors. Until now, protein-DNA interactions (PDIs) have mostly been studied in bulk without retrieving spatial information from the sample or with poor sequence resolution. New tools are needed to reveal proteins interacting with specific DNA sequences in situ for further understanding of the orchestration of transcriptional control within the nucleus. We present herein an approach to visualise individual PDIs within cells, based on the in situ proximity ligation assay (PLA). This assay, previously used for the detection of protein-protein interactions in situ, was adapted for analysis of target PDIs, using padlock probes to identify unique DNA sequences in complex genomes. As a proof-of-principle we detected histone H3 interacting with a 26bp consensus sequence of the Alu-repeat abundantly expressed in the human genome, but absent in mice. However, the mouse genome contains a highly similar sequence, providing a model system to analyse the selectivity of the developed methods. Although efficiency of detection currently is limiting, we conclude that in situ PLA can be used to achieve a highly selective analysis of PDIs in single cells.

Place, publisher, year, edition, pages
2012. Vol. 29, no 5, 589-598 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Molecular Biotechnology; Computerized Image Analysis
Identifiers
URN: urn:nbn:se:uu:diva-163960DOI: 10.1016/j.nbt.2011.08.002ISI: 000305606500012PubMedID: 21906700OAI: oai:DiVA.org:uu-163960DiVA: diva2:465845
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Carolina Wählby
Available from: 2011-12-15 Created: 2011-12-15 Last updated: 2017-12-08Bibliographically approved

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Gavrilovic, MilanLandegren, UlfWählby, CarolinaSöderberg, Ola

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Gavrilovic, MilanLandegren, UlfWählby, CarolinaSöderberg, Ola
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Molecular toolsScience for Life Laboratory, SciLifeLabComputerized Image Analysis and Human-Computer InteractionDivision of Visual Information and Interaction
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New Biotechnology
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

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