EdU Incorporation for FACS and Microscopy Analysis of DNA Replication in Budding Yeast.
DNA replication is a key determinant of chromosome segregation and stability in eukaryotes. The yeast Saccharomyces cerevisiae has been extensively used for cell cycle studies, yet simple but key parameters such as the fraction of cells in S phase in a population or the subnuclear localization of DNA synthesis have been difficult to gather for this organism. 5-ethynyl-2'-deoxyuridine (EdU) is a thymidine analogue that can be incorporated in vivo and later detected using copper-catalyzed azide alkyne cycloaddition (Click reaction) without prior DNA denaturation. This chapter describes a budding yeast strain and conditions that allow rapid EdU incorporation at moderate extracellular concentrations, followed by its efficient detection for the analysis of DNA replication in single cells by flow cytometry and fluorescence microscopy.
Institute of Molecular Genetics, CNRS UMR5535 & University Montpellier, Montpellier, France.
Nicolas
N
Talarek
Julie
J
Petit
Elisabeth
E
Gueydon
Etienne
E
Schwob
D004261:DNA Replication; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D004718:Saccharomycetales / Q000378:metabolism*; D013194:Staining and Labeling
C031086:5-ethynyl-2'-deoxyuridine; D003857:Deoxyuridine