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

Methods. 2012 Jun;57(2):149-57. doi: 10.1016/j.ymeth.2012.04.007. Epub 2012 May 3.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bromodeoxyuridine / metabolism
  • Click Chemistry
  • DNA / biosynthesis
  • DNA / chemistry
  • DNA / isolation & purification
  • DNA Replication*
  • DNA, Fungal / biosynthesis*
  • DNA, Fungal / chemistry
  • DNA, Fungal / isolation & purification
  • DNA, Single-Stranded / chemistry
  • Data Interpretation, Statistical
  • Fluorescent Antibody Technique, Indirect
  • Genome, Fungal
  • Genome, Human
  • HCT116 Cells
  • Humans
  • Hydroxyurea / pharmacology
  • Immobilized Nucleic Acids / chemistry
  • In Situ Hybridization, Fluorescence
  • Mammals
  • Nucleic Acid Synthesis Inhibitors / pharmacology
  • Saccharomyces cerevisiae / genetics
  • Staining and Labeling*
  • Statistics, Nonparametric


  • DNA, Fungal
  • DNA, Single-Stranded
  • Immobilized Nucleic Acids
  • Nucleic Acid Synthesis Inhibitors
  • DNA
  • Bromodeoxyuridine
  • Hydroxyurea