Quantifying mtDNA Heteroplasmy by Pyrosequencing

Heteroplasmy levels of C5024T (mouse) or A3243G (human) mtDNA point mutations were determined by pyrosequencing, as previously described (25 (link)). Briefly, for the C5024T mutation, a 178–base pair mtDNA fragment spanning the C5024T point mutation was PCR amplified using a biotinylated forward primer (5′-TTCCACCCTAGCTATCATAAGC-3′) and a nonbiotinylated reverse primer (5′-GTAGGTTTAATTCCTGCCAATCT-3′). For the human mutation the following primers were used: forward, nonbiotinylated 5′-CCTCCCTGTACGAAAGGACA-3′; reverse, biotinylated 5′-TGGCCATGGGTATGTTGTTA-3′. After adding Streptavidin Sepharose High-Performance beads (GE Healthcare), PCR products were purified and denatured using a PyroMark Q24 vacuum workstation (Qiagen). Sequencing was carried out with PyroMark Gold Q24 reagents and sequencer (Qiagen) according to the manufacturers’ recommendations and using the sequencing primers 5′-TGTAGGATGAAGTCTTACA-3′ (for mouse) and 5′-GGTTTGTTAAGATGGCAG-3′ (for human).