PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage.

Abstract

Because of the role that DNA damage and depletion play in human disease, it is important to develop and improve tools to assess these endpoints. This unit describes PCR-based methods to measure nuclear and mitochondrial DNA damage and copy number. Long amplicon quantitative polymerase chain reaction (LA-QPCR) is used to detect DNA damage by measuring the number of polymerase-inhibiting lesions present based on the amount of PCR amplification; real-time PCR (RT-PCR) is used to calculate genome content. In this unit, we provide step-by-step instructions to perform these assays in Homo sapiens, Mus musculus, Rattus norvegicus, Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Oryzias latipes, Fundulus grandis, and Fundulus heteroclitus, and discuss the advantages and disadvantages of these assays.

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Citation

Published Version (Please cite this version)

10.1002/0471140856.tx2011s67

Publication Info

Gonzalez-Hunt, Claudia P, John P Rooney, Ian T Ryde, Charumathi Anbalagan, Rashmi Joglekar and Joel N Meyer (2016). PCR-Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage. Curr Protoc Toxicol, 67. pp. 20.11.1–20.11.25. 10.1002/0471140856.tx2011s67 Retrieved from https://hdl.handle.net/10161/12422.

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Scholars@Duke

Gonzalez-Hunt

Claudia Gonzalez-Hunt

Research Associate, Senior

Research Interests: Mitochondrial biology, genome integrity, Parkinson's disease, neurodegeneration

Meyer

Joel Meyer

Sally Kleberg Distinguished Professorship

Dr. Meyer studies the effects of toxic agents and stressors on human and wildlife health. He is particularly interested in understanding the mechanisms by which environmental agents cause DNA damage, the molecular processes that organisms employ to protect prevent and repair DNA damage, and genetic differences that may lead to increased or decreased sensitivity to DNA damage. Mitochondrial DNA damage and repair, as well as mitochondrial function in general, are a particular focus. He studies these effects in the nematode Caenorhabditis elegans, in cell culture, and collaboratively in other laboratory model organisms as well as in human populations in the USA and globally.


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