Figure 2 Determination of m3243 A>G mutant load. m.3243A>G creates an ApaI restriction site. Polymerase chain reaction (PCR) amplification was carried out using primers A ( Rox‐tagged primer) and B (in which an artificial ApaI site was created by mismatch incorporation, as an internal digestion standard). ApaI restriction of the 258‐bp PCR product generated two (252 and 6 bp) and three (172, 80 bp and 6 bp) fragments for wild‐type and mutant species, respectively. After purification and electrophoresis using a ABI3100 genetic analyser (Applied Biosystems), digestion products were analysed by Genescan and Genotyper softwares. Only fluorescent fragments, for example, 258, 252 and 80 bp, were detected. Peak areas are indicated under the baseline. Mutant load was calculated by dividing the 80‐bp peak area by the sum of the 80‐bp and 252‐bp peak areas.