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In Vitro Replication Assay with Mammalian Cell Extracts

  • Wasia Rizwani
  • Srikumar P. Chellappan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 523)

Abstract

Regulatory mechanisms for DNA replication are crucial to the control of the cell cycle in eukaryotic cells. One of the widely used assays to understand the complex mammalian replication system is the cell-free in vitro replication assay (IVRA). 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.

Key words

Replication assay pEPI-1 plasmid nuclear and cytosolic extracts 

Notes

Acknowledgments

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

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Wasia Rizwani
    • 1
  • Srikumar P. Chellappan
    • 2
  1. 1.H. Lee Moffitt Cancer Center and Research InstituteTampaUSA
  2. 2.Drug Discovery ProgramH. Lee Moffitt Cancer Center and Research InstituteTampaUSA

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