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

研究成果: ジャーナルへの寄稿学術誌査読

84 被引用数 (Scopus)

抄録

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.

本文言語英語
ページ(範囲)149-157
ページ数9
ジャーナルMethods
57
2
DOI
出版ステータス出版済み - 6月 2012
外部発表はい

!!!All Science Journal Classification (ASJC) codes

  • 分子生物学
  • 生化学、遺伝学、分子生物学一般

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