Single-molecule analysis of DNA replication in Xenopus egg extracts

Methods. 2012 Jun;57(2):179-86. doi: 10.1016/j.ymeth.2012.03.033. Epub 2012 Apr 6.


The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Animals
  • Base Sequence
  • Cell Extracts / genetics*
  • Cell Separation
  • DNA Replication*
  • DNA, Viral / biosynthesis*
  • DNA, Viral / chemistry
  • Female
  • Flow Cytometry
  • Fluorescent Antibody Technique, Indirect
  • Immobilized Nucleic Acids / biosynthesis*
  • Immobilized Nucleic Acids / chemistry
  • Microfluidic Analytical Techniques / instrumentation
  • Oocytes / cytology*
  • Proliferating Cell Nuclear Antigen / chemistry
  • Protein Binding
  • Staining and Labeling
  • Xenopus laevis


  • Cell Extracts
  • DNA, Viral
  • Immobilized Nucleic Acids
  • Proliferating Cell Nuclear Antigen