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Combined Bidimensional Electrophoresis and Electron Microscopy to Study Specific Plasmid DNA Replication Intermediates in Human Cells

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Functional Analysis of DNA and Chromatin

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1094))

Abstract

Replication interference by specific chromosomal sequences—such as trinucleotide repeats—plays a causative, though undefined role in the aetiology of human disease, especially neurodegenerative syndromes. However, studies on these mechanisms in human cells have been hampered by poorly defined replication origins on genomic DNA. Simian Virus 40 (SV40)-based plasmids were useful in the past to overcome these experimental limits, but have been rarely amenable for the most complex and revealing molecular biology approaches to study in vivo DNA replication interference. This chapter describes a new, safe, SV40-based episomal system that replicates with very high efficiency in human cells and allows isolation of in vivo replication intermediates with high yield and purity. We describe how to use this experimental system to run preparative agarose 2D-gel and to extract specific replication intermediates to visualize by electron microscopy.

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References

  1. Fanning E, Zhao K (2009) SV40 DNA replication: from the A gene to a nanomachine. Virology 384:352–359

    Article  PubMed  CAS  Google Scholar 

  2. Follonier C, Oehler J, Herrador R, Lopes M (2013) Friedreich’s Ataxia associated GAA repeats induce replication fork reversal and unusual molecular junctions in human cells. Nat Struct Mol Biol 20:486–494

    Article  PubMed  CAS  Google Scholar 

  3. Hirt B (1967) Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol 26:365–369

    Article  PubMed  CAS  Google Scholar 

  4. Neelsen KJ et al (2013) Visualization and interpretation of eukaryotic DNA replication intermediates In Vivo by Electron Microscopy. Methods Mol Biol 1094:177–208

    Google Scholar 

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Follonier, C., Lopes, M. (2014). Combined Bidimensional Electrophoresis and Electron Microscopy to Study Specific Plasmid DNA Replication Intermediates in Human Cells. In: Stockert, J., Espada, J., Blázquez-Castro, A. (eds) Functional Analysis of DNA and Chromatin. Methods in Molecular Biology, vol 1094. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-706-8_16

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  • DOI: https://doi.org/10.1007/978-1-62703-706-8_16

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-705-1

  • Online ISBN: 978-1-62703-706-8

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