The population was genotyped by primer extension of multiplex PCR products with the detection of the allele-specific extension products by matrix-associated laser desorption/ionization time of flight (MALDI-TOF; Sequenom MassARRAY, San Diego, CA). Assays were designed with Sequenom Assay Design software v2.0.7.0, resulting in five multiplex assays. The allelotyping assay was followed according to manufacturer's instructions, with modifications. At the primary PCR step, DNA was amplified under the following conditions: initial denaturation of 95°C for 15 minutes, then 30 cycles of denaturation at 95°C for 20 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 1 minute. Finally, there was a further extension at 72°C for 3 minutes before the samples were cooled and stored at 4°C. A homogeneous MassEXTEND (hME) reaction mix containing appropriate hME EXTEND mix (1× buffer with 0.225 mM d/ddNTPs), 2.7 μM MassEXTEND primer, and 0.576 U ThermoSequenase (GE Healthcare), made up to a final volume of 2 μL with anH2O, was added to each SAP-cleaned PCR product. The microplate was then thermocycled as follows: initial denaturation of 94°C for 2 minutes, then 55 cycles of denaturation at 94°C for 5 seconds, annealing at 52°C for 5 seconds, and extension at 72°C for 5 seconds before cooling to 4°C. The sample microplate and a 384 SpectroCHIP were loaded onto the deck of the Samsung Nanodispenser. 15 nL of solution from the sample microplate was transferred onto the chip, which was read by a Bruker Autoflex Mass Spectrometer system. Data was collected with the use of SpectroACQUIRE v3.3.1.3 software and visualised with the use of MassARRAY Typer v3.4 TyperAnalyzer software. The sensitivity of the MALDI-TOF MS assay for each mutation was assessed with mixed cloned mtDNA fragments in duplicate for both uniplex and multiplex reactions (Table S1, available online).