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In Vivo Analysis of mtDNA Replication at the Single Molecule Level and with High Resolution

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Mitochondrial Gene Expression

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2192))

Abstract

Single molecule analysis of replicating DNA (SMARD) is a powerful methodology that allows in vivo analysis of replicating DNA; identification of origins of replication, assessment of fork directionality, and measurement of replication fork speed. SMARD, which has been extensively used to study replication of nuclear DNA, involves incorporation of thymidine analogs to nascent DNA chains and their subsequent visualization through immune detection. Here, we adapt and fine-tune the SMARD technique to the specifics of human and mouse mitochondrial DNA. The mito-SMARD protocol allows researchers to gain in vivo insight into mitochondrial DNA (mtDNA) replication at the single molecule level and with high resolution.

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Acknowledgement

Competing Interests: Agnel Sfeir is a cofounder and shareholder in Repare Therapeutics.

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Author notes

  1. Aaron Fraser Phillips

    Present address: Department of Developmental Genetics, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA

Authors and Affiliations

  1. Department of Developmental Genetics, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, NY, USA

    Marco Tigano & Agnel Sfeir

Authors
  1. Marco Tigano
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  2. Aaron Fraser Phillips
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  3. Agnel Sfeir
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Corresponding author

Correspondence to Agnel Sfeir .

Editor information

Editors and Affiliations

  1. Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK

    Michal Minczuk

  2. Division of Molecular Metabolism, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Solna, Sweden, Institutet Laboratory, Max Planck Institute Biology of Ageing - Karolinska, Karolinska Institutet, Stockholm, Sweden

    Joanna Rorbach

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Tigano, M., Phillips, A.F., Sfeir, A. (2021). In Vivo Analysis of mtDNA Replication at the Single Molecule Level and with High Resolution. In: Minczuk, M., Rorbach, J. (eds) Mitochondrial Gene Expression. Methods in Molecular Biology, vol 2192. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0834-0_2

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  • DOI: https://doi.org/10.1007/978-1-0716-0834-0_2

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0833-3

  • Online ISBN: 978-1-0716-0834-0

  • eBook Packages: Springer Protocols

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