Development of the MitoQ assay as a real-time quantification of mitochondrial DNA in degraded samples.

Mitochondrial DNA is a reliable genetic material for estimating maternally related haplogroups and ancestries. Exploring maternal DNA inheritance is particularly useful when nuclear DNA is degraded or limited, as the copy number of mitochondrial DNA is far greater than the copy number of nuclear DNA. Normal mitochondrial DNA copy number has been estimated to 100 copies per buccal epithelial cell, 4000 copies in skeletal cells and 7000 copies in myocardial cells. This estimation is usually performed via extrapolation from the nuclear DNA quantitation. It is essential to reduce this variability and accurately quantify the exact number of copies of mitochondrial DNA, especially in compromised samples of a forensic or ancient nature. While useful, the testing of mitochondrial DNA is often long and costly and comes with limited success. The accurate quantification of mitochondrial DNA using specific quantitative PCR assays can be used to make better decisions on the downstream testing and success of amplification. As a result, this study develops a real-time assay for the quantification of mitochondrial DNA copy number and assesses its performance on a set of degraded DNA samples. The developed MitoQ assay has been shown to be highly specific to the human mitochondrial genome with no amplification of nuclear pseudogenes being observed and outperformed a previously published concordant assay. Additionally, a high sensitivity was measured to 280 copies of mitochondrial DNA. Minimal variation was observed between each replication cycle, indicating the assay to be robust and repeatable. Overall, this study presents a real-time assay that is sensitive and robust to quantifying mitochondrial DNA copy number in degraded samples. Furthermore, there is potential to incorporate the assay as an additional target in current qPCR assays which use a six-dye chemistry and provide a complete overview of a sample's quality and quantity.