DNA replication in nucleus-free Xenopus egg extracts

Methods Mol Biol. 2009;521:229-52. doi: 10.1007/978-1-60327-815-7_13.

Abstract

Extracts derived from Xenopus laevis eggs represent a powerful cell-free system to study eukaryotic DNA replication. A variation of the system allows for DNA replication not only in a cell-free environment, but also in the absence of a nucleus. In this nucleus-free system, DNA templates are licensed with High-Speed Supernatant (HSS) and then replicated with a concentrated NucleoPlasmic Extract (NPE). This method has the advantage of allowing replication of small plasmids with desired modifications and manipulation of the nuclear environment. This chapter describes the protocols needed to prepare HSS and NPE and how these extracts are used to study DNA replication.

MeSH terms

  • Animals
  • Cell Nucleus / metabolism
  • Cell-Free System
  • Chromatin / metabolism
  • Cytosol / metabolism
  • DNA Replication*
  • Female
  • In Vitro Techniques
  • Male
  • Oocytes / metabolism
  • Proteins / metabolism
  • Spermatozoa / metabolism
  • Xenopus laevis

Substances

  • Chromatin
  • Proteins