A methodology to separate single cells and single mitochondria to determine the location of heteroplasmy in mitochondrial DNA

A mixture of mutated and wild type mitochondrial DNA is referred to as a heteroplasmic population. Mitochondrial DNA heteroplasmies have been studied at the multi-cell level with some being linked to chronic symptoms of mitochondria-based diseases. However, the mechanism producing heteroplasmy is undetermined. One question is whether mitochondrial DNA heteroplasmies are present within single mitochondria. To address this issue we developed a protocol to isolate a single mitochondria from single human leukocyte cells. The cells from an HL-60 cell culture were labeled with Mitotracker Green FM and showed to contain a heteroplasmy at the cellular level (PCR and sequencing showed a 50/50 C/T heteroplasmy at nucleotide position 12071). In order to study heteroplasmy at the single mitochondria level a pulsed UV laser was used to lyse an individual cell. Mitochondria escaped from the cell and optical tweezers were used to transfer single mitochondria into a micropipette tip. Preliminary results suggest that single mitochondria also contain the heteroplasmy.