In Vitro Replication Assay with Mammalian Cell Extracts
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.
Key wordsReplication assay pEPI-1 plasmid Nuclear and cytosolic extracts
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.