Quantifying Mitochondrial DNA Mutations

Total DNA was extracted from flash-frozen tissues using phenol–chloroform and from FACS-sorted cells using the NucleoSpin Tissue XS kit (740901.50, Takara). DNA concentration was determined spectrophotometrically (BioTek Synergy H1 hybrid). Levels of the m.5024C>T mutation were determined by “last-cycle hot” PCR^{52 (link)}, wherein the last cycle of the PCR is run using radioactively labeled nucleotides. This method removes interference from heteroduplexes formed by previous melting and annealing steps by only allowing visualization of nascent amplicons. PCR amplicons were obtained with the following primers: F-5′-CCACCCTAGCTATCATAAGCACA-3′ and B-5′-AAGCAATTGATTTGCATTCAATAGATGTAGGATGAAGTCCTGCA-3′^{20 (link)}. RFLP analysis was done by digesting amplicons with PstI-HF (R3140S, New England BioLabs), which digests the WT mtDNA but not the mutant mtDNA carrying the m.5024C>T point mutation. After digestion, products were run in a 12% polyacrylamide gel and signal was detected using the Cyclone phosphor-imaging system (Perkin Elmer) and OptiQuant software Version 5.0 (Perkin Elmer)^{20 (link)}.