Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing

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 inc...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2012-06, Vol.57 (2), p.149-157
Main Authors: Bianco, Julien N., Poli, Jérôme, Saksouk, Julie, Bacal, Julien, Silva, Maria Joao, Yoshida, Kazumasa, Lin, Yea-Lih, Tourrière, Hélène, Lengronne, Armelle, Pasero, Philippe
Format: Article
Language:English
Subjects:
Animals
Bromodeoxyuridine
Bromodeoxyuridine - metabolism
Click Chemistry
Data Interpretation, Statistical
Denaturation
DNA - biosynthesis
DNA - chemistry
DNA - isolation & purification
DNA combing
DNA fibers
DNA Replication
DNA, Fungal - biosynthesis
DNA, Fungal - chemistry
DNA, Fungal - isolation & purification
DNA, Single-Stranded - chemistry
Fluorescent Antibody Technique, Indirect
Genetics
Genome, Fungal
Genome, Human
HCT116 Cells
Humans
Hydroxyurea - pharmacology
Immobilized Nucleic Acids - chemistry
In Situ Hybridization, Fluorescence
Life Sciences
Mammalian cells
Mammals
Nucleic Acid Synthesis Inhibitors - pharmacology
Nucleotides
Replication
Replication forks
Replication origins
Saccharomyces cerevisiae
Saccharomyces cerevisiae - genetics
Staining and Labeling
Statistics, Nonparametric
Thymidine
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Summary: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.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2012.04.007