Detecting mtDNA Large-Scale Deletions

The process of detecting mtDNA large-scale deletions was conducted following similar protocols used previously, albeit with slight modifications.^{10 (link)} Two divided fragments of the entire mtDNA, which are 7.8 kb and 9.3 kb, were performed by long-range PCR. For the 7.8 kb amplification, 30 cycles were used with 98°C for 10 seconds, 68°C for 30 seconds, and 72°C for 3 minutes 30 seconds, and a final elongation at 72°C for 10 minutes. For the amplification of the 9.3 kb, the cycling conditions used were 30 cycles of 98°C for 10 seconds, 68°C for 30 seconds, and 72°C for 5 minutes, with a final elongation at 72°C for 10 minutes. The reaction mixture used for both amplifications was Phusion High Fidelity (ThermoFisher Scientific, Waltham, Mass, USA) on the SureCycler 8800 Thermal Cycler (Agilent Technologies, Inc., Santa Clara, Calif, USA). The products were then checked on a 1% agarose gel in TAE buffer for 55 minutes at 75 volts.