Analysis of differential DNA damage in the mitochondrial genome employing a semi-long run real-time PCR approach

The maintenance of the mitochondrial genomic integrity is a prerequisite for proper mitochondrial function. Due to the high concentration of reactive oxygen species (ROS) generated by the oxidative phosphorylation pathway, the mitochondrial genome is highly exposed to oxidative stress leading to mitochondrial DNA injury. Accordingly, mitochondrial DNA damage was shown to be associated with ageing as well as with numerous human diseases including neurodegenerative disorders and cancer. To date, several methods have been described to detect damaged mitochondrial DNA, but those techniques are semi-quantitative and often require high amounts of genomic input DNA. We developed a rapid and quantitative method to evaluate the relative levels of damage in mitochondrial DNA by using the real time-PCR amplification of mitochondrial DNA fragments of different lengths. We investigated mitochondrial DNA damage in SH-SY5Y human neuroblastoma cells exposed to hydrogen peroxide or stressed by over-expression of the tyrosinase gene. In the past, there has been speculation about a variable vulnerability to oxidative stress along the mitochondrial genome. Our results indicate the existence of at least one mitochondrial DNA hot spot, namely the D-Loop, being more prone to ROS-derived damage.