- Comparative Quantification of Oxidative Damage in the Genome, Telomere, and mtDNA using qPCR
Oxidative stress occurs when there is an unbalanced amount of reactive oxygen species (ROS) in the cell. These ROS can cause oxidative damage in the DNA which lead to mutations that can contribute to cancer, neurodegenerative diseases, and aging. One common form of oxidative damage is 8-oxoguanine (OG) and this can be used as a marker for oxidative stress. One type of oxidative stress is the production of superoxides. These superoxides are produced in the mitochondria and get converted into hydrogen peroxide which then can be activated by reacting with iron in the cell. Because hydrogen peroxide can diffuse into other cells before it reacts with iron, oxidative stress isn’t necessarily contained within the mitochondria where most of the ROS are produced. We want to quantify OG in mitochondrial DNA targets as well as nuclear DNA targets to gain a better understanding of where in the cell is DNA most susceptible to oxidative stress. To quantify oxidative damage in DNA, we will be using a qPCR method. Bacterial Fpg, a base excision repair enzyme, can be used to detect and cleave the DNA where OG occurs which will decrease DNA amplification in qPCR. After adding bacterial Fpg to our qPCR sample, the amount that we see decreased DNA amplification will correspond to the amount of oxidative damage present in that region of DNA. We can then use this information to compare the amounts across the different DNA target regions to gain a better understanding of where oxidative stress is most felt in the cell. This will then allow further research into the mechanisms of ROS as well as where to target for repair of oxidative damage.