Journal Article

Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications

Gergely Róna, Ildikó Scheer, Kinga Nagy, Hajnalka L. Pálinkás, Gergely Tihanyi, Máté Borsos, Angéla Békési and Beáta G. Vértessy

in Nucleic Acids Research

Volume 44, issue 3, pages e28-e28
Published in print February 2016 | ISSN: 0305-1048
Published online October 2015 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gkv977

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The role of uracil in genomic DNA has been recently re-evaluated. It is now widely accepted to be a physiologically important DNA element in diverse systems from specific phages to antibody maturation and Drosophila development. Further relevant investigations would largely benefit from a novel reliable and fast method to gain quantitative and qualitative information on uracil levels in DNA both in vitro and in situ, especially since current techniques does not allow in situ cellular detection. Here, starting from a catalytically inactive uracil-DNA glycosylase protein, we have designed several uracil sensor fusion proteins. The designed constructs can be applied as molecular recognition tools that can be detected with conventional antibodies in dot-blot applications and may also serve as in situ uracil-DNA sensors in cellular techniques. Our method is verified on numerous prokaryotic and eukaryotic cellular systems. The method is easy to use and can be applied in a high-throughput manner. It does not require expensive equipment or complex know-how, facilitating its easy implementation in any basic molecular biology laboratory. Elevated genomic uracil levels from cells of diverse genetic backgrounds and/or treated with different drugs can be demonstrated also in situ, within the cell.

Journal Article.  9022 words.  Illustrated.

Subjects: Research Methods in Life Sciences

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