期刊
METHODS
卷 57, 期 2, 页码 149-157出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2012.04.007
关键词
DNA replication; DNA combing; DNA fibers; Replication origins; Replication forks
资金
- ANR
- Fondation pour la Recherche Medicale (Equipe FRM)
- ARC
- French Ministere de la Recherche et de l'Enseignement Superieur
- CNRS
- Region Languedoc-Roussillon
DNA combing is a powerful method developed by Bensimon and colleagues to stretch DNA molecules on silanized glass coverslips. This technique provides a unique way to monitor the activation of replication origins and the progression of replication forks at the level of single DNA molecules, after incorporation of thymidine analogs, such as 5-bromo-2'-deoxyuridine (BrdU), 5-iodo-2'-deoxyuridine (IdU) and 5-chloro-2'-deoxyuridine (CldU) in newly-synthesized DNA. Unlike microarray-based approaches, this assay gives access to the variability of replication profiles in individual cells. It can also be used to monitor the effect of DNA lesions on fork progression, arrest and restart. In this review, we propose standard DNA combing methods to analyze DNA replication in budding yeast and in human cells. We also show that 5-ethynyl-2'-deoxyuridine (EdU) can be used as a good alternative to BrdU for DNA combing analysis, as unlike halogenated nucleotides, it can be detected without prior denaturation of DNA. (c) 2012 Elsevier Inc. All rights reserved.
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