title: Quantification of DNA Damage by Real-Time qPCR.
abstract: This chapter describes the use of real-time qPCR to quantify damages in genomic DNA. The method is based on the ability of a lesion in one strand to inhibit restriction enzyme digestion of double-stranded DNA. Subsequent amplification of the complementary strand after restriction cleavage gives a quantitative measure of the damage content in that site (Real-time qPCR Analysis of Damage Frequency; RADF). We compare the RADF assay with the commonly used technique to assess damages by their ability to inhibit amplification of a large PCR fragment relative to a short PCR fragment. The RADF method described here is quick, accurate and allows the detection of nuclear and mitochondrial DNA damage in detailed regions.
authors.affiliation: Department of Medical Biochemistry, Institute of Clinical Medicine, Oslo University Hospital and University of Oslo, Sognsvannsveien 20, 0027, Oslo, Norway.|Department of Microbiology, Oslo University Hospital and University of Oslo, Oslo, Norway.
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Medical Biochemistry, Institute of Clinical Medicine, Oslo University Hospital and University of Oslo, Sognsvannsveien 20, 0027, Oslo, Norway.|Department of Microbiology, Oslo University Hospital and University of Oslo, Oslo, Norway.
authors.forename: Katja
authors.initials: K
authors.lastname: Scheffler
authors.affiliation: Institute of Clinical Epidemiology and Molecular Biology, Akershus University Hospital and University of Oslo, Lørenskog, Norway.
authors.forename: Ying
authors.initials: Y
authors.lastname: Esbensen
authors.affiliation: Department of Medical Biochemistry, Institute of Clinical Medicine, Oslo University Hospital and University of Oslo, Sognsvannsveien 20, 0027, Oslo, Norway. lars.eide@medisin.uio.no.
authors.forename: Lars
authors.initials: L
authors.lastname: Eide
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004249:DNA Damage / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004272:DNA, Mitochondrial / Q000032:analysis* / Q000235:genetics; D006801:Humans; D008928:Mitochondria / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; D004272:DNA, Mitochondrial; D017382:Reactive Oxygen Species
keywords: DNA damage; DNA repair; mtDNA