Open AccessPCR‐Based Analysis of Mitochondrial DNA Copy Number, Mitochondrial DNA Damage, and Nuclear DNA Damage
Author(s) -
GonzalezHunt Claudia P.,
Rooney John P.,
Ryde Ian T.,
Anbalagan Charumathi,
Joglekar Rashmi,
Meyer Joel N.
Publication year - 2016
Publication title -
current protocols in toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.449
H-Index - 23
eISSN - 1934-9262
pISSN - 1934-9254
DOI - 10.1002/0471140856.tx2011s67
Subject(s) - mitochondrial dna , biology , amplicon , nuclear dna , polymerase chain reaction , microbiology and biotechnology , dna damage , real time polymerase chain reaction , caenorhabditis elegans , dna , digital polymerase chain reaction , in silico pcr , genetics , applications of pcr , gene , multiplex polymerase chain reaction
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. © 2016 by John Wiley & Sons, Inc.