A Droplet Digital Polymerase Chain Reaction Assay for Detecting and Quantifying Pre-Existing Mutations and De Novo...

Development of mitochondria-targeted base editors as a gene therapy strategy for mitochondrial deoxyribonucleic acid (mtDNA)-related diseases, such as MT-ATP6 Leigh syndrome (LS), requires using in vitro models for proof-of-concept evaluations. Here, we developed a droplet digital polymerase chain reaction (ddPCR)-based assay that uses detection probes specific to wild-type, mutant, or edited mtDNA alleles to determine the heteroplasmy level in cells carrying different mtDNA variants. We used a pathogenic human MT-ATP6 allele as an example to demonstrate the development and optimization pipeline of this assay. We optimized assay parameters and designed experiments to validate the precision of the assay in quantifying MT-ATP6 variants and their fraction among the total mtDNA copy number. In addition, our data indicated that this assay is highly sensitive. To allow high-throughput screening, we then developed an R-based analysis pipeline to streamline the analysis workflow for determining MT-ATP6 variant fractional abundance. Our data demonstrated that the assay achieved the high resolution required to differentiate various heteroplasmic MT-ATP6 alleles. Future studies can build upon the current protocol and develop similar ddPCR assays to perform high-throughput screening for the identification of cell lines or tissue samples with specified mtDNA alleles.