Talarek Nicolas, Petit Julie, Gueydon Elisabeth, Schwob Etienne
Institute of Molecular Genetics, CNRS UMR5535 & University Montpellier, Montpellier, France.
Methods Mol Biol. 2015;1300:105-12. doi: 10.1007/978-1-4939-2596-4_7.
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.
DNA复制是真核生物中染色体分离和稳定性的关键决定因素。酿酒酵母已被广泛用于细胞周期研究，然而，对于这种生物体来说，一些简单但关键的参数，如群体中处于S期的细胞比例或DNA合成的亚核定位，却很难收集到。5-乙炔基-2'-脱氧尿苷（EdU）是一种胸腺嘧啶类似物，它可以在体内掺入，随后无需事先进行DNA变性，即可使用铜催化的叠氮化物炔烃环加成反应（点击反应）进行检测。本章介绍了一种芽殖酵母菌株及其培养条件，这些条件可使细胞在中等细胞外浓度下快速掺入EdU，随后对其进行有效检测，以便通过流式细胞术和荧光显微镜分析单细胞中的DNA复制情况。
