Positive calls were confirmed in an independent aliquot from the original DNA sample with the use of last-cycle fluorescent PCR-RFLP as described previously.15 This also established the percentage mutated mtDNA. For the m.1555A→G mutation, heteroplasmy was quantified by cloning and sequencing of 23 independent mtDNA fragments from an independent aliquot from the original DNA sample. Primers and restriction enzymes and digests are shown in Table S2.