BrdU Incorporation and Labeling of Nascent DNA to Investigate Archaeal Replication Using Super-Resolution Imaging

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

Abstract

The labeling and specific detection of nascent DNA by the incorporation of thymidine analogs provide crucial information about DNA replication dynamics without requiring the intracellular expression of fluorescent proteins. After cell fixation and permeabilization, specific detection of thymidine analogs by antibodies can be performed using super-resolution imaging techniques. Here we describe a protocol to label nascent DNA using 5′-bromo-2′-deoxyuridine (BrdU) in Haloferax volcanii cells and generate super-resolved images of neo-synthesized DNA foci either by 3D Structured illumination microscopy (3D-SIM) or Stochastic Optical Reconstruction Microscopy (STORM).

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages419-434
Number of pages16
DOIs
Publication statusPublished - 1 Jan 2022

Publication series

NameMethods in Molecular Biology
Volume2522
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • 3D structured illuminated microscopy (3D-SIM)
  • Archaea
  • BrdU
  • Haloferax volcanii
  • Nascent DNA labeling
  • Replication
  • Stochastic optical reconstruction microscopy (STORM)
  • Super-resolution imaging

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