Unit

UNIT 22.10 Replication Labeling with Halogenated Thymidine Analogs

  1. Tomoki Yokochi,
  2. David M. Gilbert

Published Online: 1 JUN 2007

DOI: 10.1002/0471143030.cb2210s35

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Yokochi, T. and Gilbert, D. M. 2007. Replication Labeling with Halogenated Thymidine Analogs. Current Protocols in Cell Biology. 35:22.10:22.10.1–22.10.14.

Author Information

  1. Department of Biological Science Florida State University Tallahassee, Florida

Publication History

  1. Published Online: 1 JUN 2007
  2. Published Print: JUN 2007

Abstract

In this unit, several basic protocols to identify sites of DNA replication utilizing incorporation of halogenated thymidine analogs into DNA, followed by immunofluorescent imaging are described. Antibodies specific for halogenated thymidine analogs such as bromodeoxyuridine (BrdU), chlorodeoxyuridine (CldU), and iododeoxyuridine (IdU) can provide a rapid, nonhazardous, and sensitive method for detecting DNA replication in single cells, in a manner analogous to the traditional use of tritiated thymidine. In combination with different techniques to prepare the DNA template, a variety of DNA replication–related events can be examined by conventional fluorescence-microscopic approaches. Because origin firing and the progression of replication forks are regulated in the context of subnuclear compartments through protein-protein interactions, chromatin modifications, and subnuclear localization of replication clusters, visualizing replication foci significantly facilitates understanding of nuclear dynamics during S-phase.

Keywords:

  • DNA replication;
  • mammalian cells;
  • 5-Bromo-2′-deoxyuridine (BrdU);
  • halogenated thymidine analogs;
  • DNA fiber