Abstract
It is imperative that dividing cells maintain replication fork integrity in order to prevent DNA damage and cell death. The investigation of DNA replication is of high importance as alterations in this process can lead to genomic instability, a known causative factor of tumor development. A simple, sensitive, and informative technique which enables the study of DNA replication, is the DNA fiber assay, an adaptation of which is described in this chapter. The DNA fiber method is a powerful tool, which allows the quantitative and qualitative analysis of DNA replication at the single molecule level. The sequential pulse labeling of live cells with two thymidine analogues and the subsequent detection with specific antibodies and fluorescence imaging allows direct examination of sites of DNA synthesis. In this chapter, we describe how this assay can be performed in conditions of low oxygen levels (hypoxia)—a physiologically relevant stress that occurs in most solid tumors. Moreover, we suggest ways on how to overcome the technical problems that arise while using the hypoxic chambers.
The authors disclose no potential conflicts of interest.
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Acknowledgments
We thank all members of the Hammond lab, past and present, for their assistance in the development of this protocol. DB and EMH are supported by Cancer Research UK (grant awarded to EMH). This work was supported by Cancer Research UK (CR-UK) grant number C38302/A12981, through a Cancer Research UK Oxford Centre Prize DPhil Studentship (awarded to IPF).
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Foskolou, I.P., Biasoli, D., Olcina, M.M., Hammond, E.M. (2016). Measuring DNA Replication in Hypoxic Conditions. In: Koumenis, C., Coussens, L., Giaccia, A., Hammond, E. (eds) Tumor Microenvironment. Advances in Experimental Medicine and Biology, vol 899. Springer, Cham. https://doi.org/10.1007/978-3-319-26666-4_2
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DOI: https://doi.org/10.1007/978-3-319-26666-4_2
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