UNIT 22.10 Replication Labeling with Halogenated Thymidine Analogs
Published Online: 1 JUN 2007
Copyright © 2007 by John Wiley and Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 JUN 2007
- Published Print: JUN 2007
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.
- DNA replication;
- mammalian cells;
- 5-Bromo-2′-deoxyuridine (BrdU);
- halogenated thymidine analogs;
- DNA fiber