Quantification of DNA damage and repair in mitochondrial, nuclear, and bacterial genomes by real-time PCR

Abstract

DNA damage caused by genotoxic insults is often used as an indicator of specific diseases, environmental challenges, and metabolic processes. To date, various different methods have been described to detect damaged DNA. Many techniques need high amounts of DNA for the analysis and/or require the exact determination of DNA template concentration. Here, we describe a rapid and quantitative method for the evaluation of the relative levels of damage in mitochondrial, nuclear, and bacterial DNA in comparison to untreated controls. The approach is based on the real-time PCR amplification of DNA fragments of two different lengths in the respective samples. DNA damage detection using this protocol is gene-specific. The technique can also be expanded to monitor DNA repair and to detect genomic hot-spots for DNA lesions.