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

Julien N. Bianco, Jérôme Poli, Julie Saksouk, Julien Bacal, Maria Joao Silva, Kazumasa Yoshida, Yea Lih Lin, Hélène Tourrière, Armelle Lengronne, Philippe Pasero

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)149-157
Number of pages9
JournalMethods
Volume57
Issue number2
DOIs
Publication statusPublished - Jun 1 2012
Externally publishedYes

Fingerprint

Saccharomycetales
DNA Replication
Yeast
Cells
DNA
Replication Origin
Nucleic Acid Denaturation
Idoxuridine
Deoxyuridine
Bromodeoxyuridine
Denaturation
Molecules
Thymidine
Glass
Microarrays
Nucleotides
Assays
Chemical activation

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Bianco, J. N., Poli, J., Saksouk, J., Bacal, J., Silva, M. J., Yoshida, K., ... Pasero, P. (2012). Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing. Methods, 57(2), 149-157. https://doi.org/10.1016/j.ymeth.2012.04.007

Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing. / Bianco, Julien N.; Poli, Jérôme; Saksouk, Julie; Bacal, Julien; Silva, Maria Joao; Yoshida, Kazumasa; Lin, Yea Lih; Tourrière, Hélène; Lengronne, Armelle; Pasero, Philippe.

In: Methods, Vol. 57, No. 2, 01.06.2012, p. 149-157.

Research output: Contribution to journalArticle

Bianco, JN, Poli, J, Saksouk, J, Bacal, J, Silva, MJ, Yoshida, K, Lin, YL, Tourrière, H, Lengronne, A & Pasero, P 2012, 'Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing', Methods, vol. 57, no. 2, pp. 149-157. https://doi.org/10.1016/j.ymeth.2012.04.007
Bianco, Julien N. ; Poli, Jérôme ; Saksouk, Julie ; Bacal, Julien ; Silva, Maria Joao ; Yoshida, Kazumasa ; Lin, Yea Lih ; Tourrière, Hélène ; Lengronne, Armelle ; Pasero, Philippe. / Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing. In: Methods. 2012 ; Vol. 57, No. 2. pp. 149-157.
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