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In Vitro Replication Assay with Mammalian Cell Extracts

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Chromatin Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1288))

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Abstract

Regulatory mechanisms are crucial to control DNA replication during cell cycle in eukaryotic cells. Cell-free in vitro replication assay (IVRA) is one of the widely used assays to understand the complex mammalian replication system. IVRA can provide a snapshot of the regulatory mechanisms controlling replication in higher eukaryotes by using a single plasmid, pEPI-1. This chapter outlines the general strategies and protocols used to perform IVRA to study the differential recruitment of replication factors either independently or in combination, based on the experience in studying the role of prohibitin in replication as well as other published protocols. This method can be employed to identify not only proteins that assist replication but also proteins that inhibit replication of mammalian genome.

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Acknowledgements

Work in the Chellappan lab was supported by the grants CA63136, CA77301, CA139612, and CA127725 from the NIH. We wish to thank Mark Alexandrow, Moffitt Cancer Center, Tampa, for providing the pEPI-1 plasmid and for helpful discussions.

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Authors and Affiliations

  1. Department of Biochemistry, Osmania University, Hyderabad, Telangana, 500 007, India

    Wasia Rizwani

  2. Department of Tumor Biology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia drive, SRB-4th floor, Tampa, FL, 33612, USA

    Srikumar P. Chellappan Ph.D.

Authors
  1. Wasia Rizwani
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  2. Srikumar P. Chellappan Ph.D.
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Corresponding author

Correspondence to Srikumar P. Chellappan Ph.D. .

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Editors and Affiliations

  1. H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida, USA

    Srikumar P. Chellappan

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Rizwani, W., Chellappan, S.P. (2015). In Vitro Replication Assay with Mammalian Cell Extracts. In: Chellappan, S. (eds) Chromatin Protocols. Methods in Molecular Biology, vol 1288. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2474-5_20

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  • DOI: https://doi.org/10.1007/978-1-4939-2474-5_20

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2473-8

  • Online ISBN: 978-1-4939-2474-5

  • eBook Packages: Springer Protocols

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