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Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry
We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985630/ https://www.ncbi.nlm.nih.gov/pubmed/24452797 http://dx.doi.org/10.1093/nar/gkt1406 |
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author | Vranken, Charlotte Deen, Jochem Dirix, Lieve Stakenborg, Tim Dehaen, Wim Leen, Volker Hofkens, Johan Neely, Robert K. |
author_facet | Vranken, Charlotte Deen, Jochem Dirix, Lieve Stakenborg, Tim Dehaen, Wim Leen, Volker Hofkens, Johan Neely, Robert K. |
author_sort | Vranken, Charlotte |
collection | PubMed |
description | We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes transalkylate DNA with the cofactor we tested (a readily prepared s-adenosyl-l-methionine analogue). |
format | Online Article Text |
id | pubmed-3985630 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-39856302014-04-18 Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry Vranken, Charlotte Deen, Jochem Dirix, Lieve Stakenborg, Tim Dehaen, Wim Leen, Volker Hofkens, Johan Neely, Robert K. Nucleic Acids Res Methods Online We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes transalkylate DNA with the cofactor we tested (a readily prepared s-adenosyl-l-methionine analogue). Oxford University Press 2014-04 2014-01-21 /pmc/articles/PMC3985630/ /pubmed/24452797 http://dx.doi.org/10.1093/nar/gkt1406 Text en © The Author(s) 2014. Published by Oxford University Press. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Vranken, Charlotte Deen, Jochem Dirix, Lieve Stakenborg, Tim Dehaen, Wim Leen, Volker Hofkens, Johan Neely, Robert K. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry |
title | Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry |
title_full | Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry |
title_fullStr | Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry |
title_full_unstemmed | Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry |
title_short | Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry |
title_sort | super-resolution optical dna mapping via dna methyltransferase-directed click chemistry |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985630/ https://www.ncbi.nlm.nih.gov/pubmed/24452797 http://dx.doi.org/10.1093/nar/gkt1406 |
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