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Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications
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 reli...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756853/ https://www.ncbi.nlm.nih.gov/pubmed/26429970 http://dx.doi.org/10.1093/nar/gkv977 |
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author | Róna, Gergely Scheer, Ildikó Nagy, Kinga Pálinkás, Hajnalka L. Tihanyi, Gergely Borsos, Máté Békési, Angéla Vértessy, Beáta G. |
author_facet | Róna, Gergely Scheer, Ildikó Nagy, Kinga Pálinkás, Hajnalka L. Tihanyi, Gergely Borsos, Máté Békési, Angéla Vértessy, Beáta G. |
author_sort | Róna, Gergely |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4756853 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-47568532016-02-18 Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications Róna, Gergely Scheer, Ildikó Nagy, Kinga Pálinkás, Hajnalka L. Tihanyi, Gergely Borsos, Máté Békési, Angéla Vértessy, Beáta G. Nucleic Acids Res Methods Online 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. Oxford University Press 2016-02-18 2015-10-01 /pmc/articles/PMC4756853/ /pubmed/26429970 http://dx.doi.org/10.1093/nar/gkv977 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Róna, Gergely Scheer, Ildikó Nagy, Kinga Pálinkás, Hajnalka L. Tihanyi, Gergely Borsos, Máté Békési, Angéla Vértessy, Beáta G. Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications |
title | Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications |
title_full | Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications |
title_fullStr | Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications |
title_full_unstemmed | Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications |
title_short | Detection of uracil within DNA using a sensitive labeling method for in vitro and cellular applications |
title_sort | detection of uracil within dna using a sensitive labeling method for in vitro and cellular applications |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756853/ https://www.ncbi.nlm.nih.gov/pubmed/26429970 http://dx.doi.org/10.1093/nar/gkv977 |
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