Density transfer as a method to analyze the progression of DNA replication forks

Methods Mol Biol. 2009;521:203-13. doi: 10.1007/978-1-60327-815-7_11.

Abstract

The density transfer technique is a valuable tool to examine the progression of individual DNA replication forks. It is based on the transfer of cells from a medium containing dense isotopes to a medium with light (normal) isotopes (or vice versa), to obtain DNA sequences hybrid in density that can be identified as replicated molecules. Using specific DNA probes along a chromosome, the dense isotope transfer method allows determining the extent of replication at any position of a replicon and the rate of replication fork progression. In the eukaryotic model budding yeast, this technique has been useful to establish a role for different proteins during the elongation of chromosomal replication and to analyze the movement and stability of DNA replication forks under different experimental conditions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carbon Isotopes
  • Centrifugation, Density Gradient
  • Chromosomes, Fungal / metabolism
  • Culture Media
  • DNA Probes
  • DNA Replication*
  • DNA, Fungal / biosynthesis*
  • DNA, Fungal / genetics
  • DNA, Fungal / isolation & purification*
  • Nitrogen Isotopes
  • Nucleic Acid Hybridization
  • Replicon
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • Carbon Isotopes
  • Culture Media
  • DNA Probes
  • DNA, Fungal
  • Nitrogen Isotopes
  • Saccharomyces cerevisiae Proteins