Droplet digital polymerase chain reaction to measure heteroplasmic m.3243A>G mitochondrial mutations

Different mitochondrial DNA genotypes can coexist in a cell population as well as in a single cell, a condition known as heteroplasmy. Here, we accurately determined the heteroplasmy levels of the m.3243A>G mutation, which is the most frequently identified mutation in patients with mitochondrial diseases, using droplet digital polymerase chain reaction (ddPCR).

The m.3243A>G heteroplasmy levels in artificial heteroplasmy controls mixed with various proportions of wild-type and mutant plasmids were measured using ddPCR, PCR-restriction fragment length polymorphism, and Sanger sequencing. The m.3243A>G heteroplasmy levels in DNA, extracted from the peripheral blood of patients with suspected mitochondrial disease and healthy subjects, were determined using ddPCR.

The accuracy of the ddPCR method was high. The lower limit of detection was 0.1%, which indicated its higher sensitivity compared with other methods. The m.3243A>G heteroplasmy levels in peripheral blood, measured using ddPCR, correlated inversely with age at the time of analysis. The m.3243A>G mutation may be overlooked in the peripheral blood-derived DNA of elderly people, as patients >60 years of age have heteroplasmy levels <10%, which is difficult to detect using methods other than the highly sensitive ddPCR.

ddPCR may be considered an accurate and sensitive method for measuring m.3243 A>G heteroplasmy levels of mitochondrial DNA.