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DNA Replication pp 43–65Cite as

High-Resolution Analysis of Mammalian DNA Replication Units

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1300))

Abstract

Genomic DNA of a eukaryotic cell is replicated once during the S-phase of the cell cycle to precisely maintain the complete genetic information. In the course of S-phase, semiconservative DNA synthesis is sequentially initiated and performed at thousands of discrete patches of the DNA helix termed replicons. At any given moment of S-phase, multiple replicons are active in parallel in different parts of the genome. In the last decades, tools and methods to visualize DNA synthesis inside cells have been developed. Pulse labeling with nucleotides as well as detecting components of the replication machinery yielded an overall picture of multiple discrete sites of active DNA synthesis termed replication foci (RFi) and forming spatiotemporal patterns within the cell nucleus. Recent advances in fluorescence microscopy and digital imaging in combination with computational image analysis allow a comprehensive quantitative analysis of RFi and provide valuable insights into the organization of the genomic DNA replication process and also of the genome itself. In this chapter, we describe in detail protocols for the visualization and quantification of RFi at different levels of optical and physical resolution.

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Acknowledgements

We thank all present and past members of the laboratory for their contributions over the years. Vadim O. Chagin was supported by grants of the Russian Foundation for Basic Research (## 12-04-01489a, 13-04-00442a). The laboratory of M. Cristina Cardoso is supported by grants of the German Research Foundation (DFG CA 198/9) and the Federal Ministry of Education and Research (BMBF).

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    Authors and Affiliations

    1. Institute of Cytology, Russian Academy of Science, Saint Petersburg, Russia

      Vadim O. Chagin

    2. Cell Biology and Epigenetics, Department of Biology, Technische Universität Darmstadt, Schnittspahnstr. 10, 64287, Darmstadt, Germany

      Marius Reinhart & M. Cristina Cardoso

    Authors
    1. Vadim O. Chagin
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    2. Marius Reinhart
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    3. M. Cristina Cardoso
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    Corresponding author

    Correspondence to M. Cristina Cardoso .

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    Editors and Affiliations

    1. Coventry, United Kingdom

      Sonya Vengrova

    2. Warwick Medical School, University of Warwick, Coventry, United Kingdom

      Jacob Dalgaard

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    Chagin, V.O., Reinhart, M., Cardoso, M.C. (2015). High-Resolution Analysis of Mammalian DNA Replication Units. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 1300. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2596-4_3

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    • DOI: https://doi.org/10.1007/978-1-4939-2596-4_3

    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-2595-7

    • Online ISBN: 978-1-4939-2596-4

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