Replication labeling with halogenated thymidine analogs.

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.