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Nucleic Acids Res. 2016 Feb 18;44(3):e28. doi: 10.1093/nar/gkv977. Epub 2015 Oct 1.

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

Author information

1
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Magyar Tudósok Str. 2, H-1117 Budapest, Hungary Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Szt Gellért Square 4, H-1111 Budapest, Hungary rona.gergely@ttk.mta.hu.
2
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Magyar Tudósok Str. 2, H-1117 Budapest, Hungary Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Szt Gellért Square 4, H-1111 Budapest, Hungary.
3
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Magyar Tudósok Str. 2, H-1117 Budapest, Hungary Doctoral School of Multidisciplinary Medical Science, University of Szeged, H-6720 Szeged, Hungary.
4
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Magyar Tudósok Str. 2, H-1117 Budapest, Hungary.
5
Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Magyar Tudósok Str. 2, H-1117 Budapest, Hungary Department of Applied Biotechnology and Food Sciences, Budapest University of Technology and Economics, Szt Gellért Square 4, H-1111 Budapest, Hungary vertessy@mail.bme.hu.

Abstract

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.

PMID:
26429970
PMCID:
PMC4756853
DOI:
10.1093/nar/gkv977
[Indexed for MEDLINE]
Free PMC Article

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