Analysis of Replicating Mitochondrial DNA by In Organello Labeling and Two-Dimensional Agarose Gel Electrophoresis

  • Ian J. HoltEmail author
  • Lawrence Kazak
  • Aurelio Reyes
  • Stuart R. Wood
Part of the Methods in Molecular Biology book series (MIMB, volume 1351)


Our understanding of the mechanisms of DNA replication in a broad range of organisms and viruses has benefited from the application of two-dimensional agarose gel electrophoresis (2D-AGE). The method resolves DNA molecules on the basis of size and shape and is technically straightforward. 2D-AGE sparked controversy in the field of mitochondria when it revealed replicating molecules with lengthy tracts of RNA, a phenomenon never before reported in nature. More recently, radioisotope labeling of the DNA in the mitochondria has been coupled with 2D-AGE. In its first application, this procedure helped to delineate the “bootlace mechanism of mitochondrial DNA replication,” in which processed mitochondrial transcripts are hybridized to the lagging strand template at the replication fork as the leading DNA strand is synthesized. This chapter provides details of the method, how it has been applied to date and concludes with some potential future applications of the technique.

Key words

DNA replication DNA labeling Mitochondrial DNA Two-dimensional agarose gel electrophoresis 2D-AGE Replication intermediates Humans Mammals 



We thank Drs. Pohjoismäki and Jacobs for the EM image of the replicating mitochondrial DNA molecule, and the Medical Research Council for funding.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Ian J. Holt
    • 1
    Email author
  • Lawrence Kazak
    • 2
  • Aurelio Reyes
    • 2
  • Stuart R. Wood
    • 2
  1. 1.MRC-National Institute for Medical ResearchLondonUK
  2. 2.MRC-Mitochondrial Biology UnitCambridgeUK

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