title: 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.
authors.affiliation: Nicholas School of the Environment, Duke University, Durham, North Carolina.
authors.forename: Claudia P
authors.initials: CP
authors.lastname: Gonzalez-Hunt
authors.affiliation: Nicholas School of the Environment, Duke University, Durham, North Carolina.
authors.forename: John P
authors.initials: JP
authors.lastname: Rooney
authors.affiliation: Nicholas School of the Environment, Duke University, Durham, North Carolina.
authors.forename: Ian T
authors.initials: IT
authors.lastname: Ryde
authors.affiliation: Nicholas School of the Environment, Duke University, Durham, North Carolina.
authors.forename: Charumathi
authors.initials: C
authors.lastname: Anbalagan
authors.affiliation: Nicholas School of the Environment, Duke University, Durham, North Carolina.
authors.forename: Rashmi
authors.initials: R
authors.lastname: Joglekar
authors.affiliation: Nicholas School of the Environment, Duke University, Durham, North Carolina.
authors.forename: Joel N
authors.initials: JN
authors.lastname: Meyer
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000235:genetics*; D056915:DNA Copy Number Variations / Q000235:genetics*; D004249:DNA Damage; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial
keywords: C. elegans; DNA damage; mitochondrial DNA; mitochondrial DNA copy number; qPCR
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