DNA Replication pp 673-687

Part of the Methods in Molecular Biology book series (MIMB, volume 521)

Use of DNA Combing for Studying DNA Replication In Vivo in Yeast and Mammalian Cells

  • Etienne Schwob
  • Christelle de Renty
  • Vincent Coulon
  • Thierry Gostan
  • Cécile Boyer
  • Linda Camet-Gabut
  • Claire Amato
Protocol

Summary

Plasticity is an inherent feature of chromosomal DNA replication in eukaryotes. Potential origins of DNA replication are made in excess, but are used (fired) in a partly stochastic, partly programmed manner throughout the S phase of the cell cycle. Since most origins have a firing efficiency below 50%, population-based analysis methods yield a cumulative picture of origin activity (obtained by accretion) that does not accurately describe how chromosomes are replicated in single cells. DNA combing is a method that allows the alignment on silanized glass coverslips, at high density and with uniform stretching, of single DNA molecules in the Mb range. If this DNA is isolated from cells that have been labelled with halogenated nucleotides (BrdU, CldU, IdU), it is possible to determine the density and position of replication origins as well as the rate and symmetry of fork progression, quantitatively and on single DNA molecules. This chapter will successively describe (a) the preparation of silanized coverslips, (b) the incorporation of halogenated nucleotides in newly synthesized DNA in yeast and mammalian cell lines, (c) the preparation and combing of genomic DNA, and finally (d) the acquisition and analysis of single-molecule images to extract salient features of replication dynamics.

Key words

DNA combing Single molecule Silanization BrdU CldU/IdU Mouse embryonic fibroblasts DNA fibre immuno-fluorescence Automated fibre detection and analysis Inter-origin distance Replication fork speed 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Etienne Schwob
    • 1
  • Christelle de Renty
    • 1
  • Vincent Coulon
    • 1
  • Thierry Gostan
    • 1
  • Cécile Boyer
    • 1
  • Linda Camet-Gabut
    • 1
  • Claire Amato
    • 1
  1. 1.Institut de Génétique Moléculaire de Montpellier (IGMM)CNRS UMR5535 & IFR122, 1919 route de MendeFrance

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