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Molecular Combing of Single DNA Molecules on the 10 Megabase Scale

DNA combing allows the investigation of DNA replication on genomic single DNA molecules, but the lengths that can be analysed have been restricted to molecules of 200–500 kb. We have improved the DNA combing procedure so that DNA molecules can be analysed up to the length of entire chromosomes in fi...

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Autores principales: Kaykov, Atanas, Taillefumier, Thibaud, Bensimon, Aaron, Nurse, Paul
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726065/
https://www.ncbi.nlm.nih.gov/pubmed/26781994
http://dx.doi.org/10.1038/srep19636
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author Kaykov, Atanas
Taillefumier, Thibaud
Bensimon, Aaron
Nurse, Paul
author_facet Kaykov, Atanas
Taillefumier, Thibaud
Bensimon, Aaron
Nurse, Paul
author_sort Kaykov, Atanas
collection PubMed
description DNA combing allows the investigation of DNA replication on genomic single DNA molecules, but the lengths that can be analysed have been restricted to molecules of 200–500 kb. We have improved the DNA combing procedure so that DNA molecules can be analysed up to the length of entire chromosomes in fission yeast and up to 12 Mb fragments in human cells. Combing multi-Mb-scale DNA molecules revealed previously undetected origin clusters in fission yeast and shows that in human cells replication origins fire stochastically forming clusters of fired origins with an average size of 370 kb. We estimate that a single human cell forms around 3200 clusters at mid S-phase and fires approximately 100,000 origins to complete genome duplication. The procedure presented here will be adaptable to other organisms and experimental conditions.
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spelling pubmed-47260652016-01-27 Molecular Combing of Single DNA Molecules on the 10 Megabase Scale Kaykov, Atanas Taillefumier, Thibaud Bensimon, Aaron Nurse, Paul Sci Rep Article DNA combing allows the investigation of DNA replication on genomic single DNA molecules, but the lengths that can be analysed have been restricted to molecules of 200–500 kb. We have improved the DNA combing procedure so that DNA molecules can be analysed up to the length of entire chromosomes in fission yeast and up to 12 Mb fragments in human cells. Combing multi-Mb-scale DNA molecules revealed previously undetected origin clusters in fission yeast and shows that in human cells replication origins fire stochastically forming clusters of fired origins with an average size of 370 kb. We estimate that a single human cell forms around 3200 clusters at mid S-phase and fires approximately 100,000 origins to complete genome duplication. The procedure presented here will be adaptable to other organisms and experimental conditions. Nature Publishing Group 2016-01-19 /pmc/articles/PMC4726065/ /pubmed/26781994 http://dx.doi.org/10.1038/srep19636 Text en Copyright © 2016, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Kaykov, Atanas
Taillefumier, Thibaud
Bensimon, Aaron
Nurse, Paul
Molecular Combing of Single DNA Molecules on the 10 Megabase Scale
title Molecular Combing of Single DNA Molecules on the 10 Megabase Scale
title_full Molecular Combing of Single DNA Molecules on the 10 Megabase Scale
title_fullStr Molecular Combing of Single DNA Molecules on the 10 Megabase Scale
title_full_unstemmed Molecular Combing of Single DNA Molecules on the 10 Megabase Scale
title_short Molecular Combing of Single DNA Molecules on the 10 Megabase Scale
title_sort molecular combing of single dna molecules on the 10 megabase scale
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726065/
https://www.ncbi.nlm.nih.gov/pubmed/26781994
http://dx.doi.org/10.1038/srep19636
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