Next‐Generation Sequencing to Characterize Mitochondrial Genomic DNA Heteroplasmy

Abstract

Mitochondria play a very important role in many crucial cellular functions. Each eukaryotic cell contains hundreds of mitochondria with hundreds of mitochondrial genomes. Mutant and wild-type mitochondrial DNA (mtDNA) may co-exist as heteroplasmy and cause human disease. The purpose of the protocols in this article is to simultaneously determine the mtDNA sequence and quantify the heteroplasmy level using parallel sequencing. The protocols include mitochondrial genomic DNA PCR amplification of two full-length products using two distinct sets of PCR primers. The PCR products are mixed at an equimolar ratio, and the samples are then barcoded and sequenced with highthroughput next-generation sequencing technology. This technology is highly sensitive, specific, and accurate in determining mtDNA mutations and the degree/level of heteroplasmy.

Common methods in mitochondrial research (Review)

“…PcR, as a molecular biology technology that emerged in the 1980s, is a method for enzymatically synthesizing and amplifying specific nucleic acid fragments in vitro based on the semi-conservative replication mechanism of dNA. This can purposefully amplify target regions and is especially suitable for enriching small-scale genomes such as mtdNA (349)(350)(351)(352)(353). However, mtdNA is present in primer-binding regions, but accuracy is not sufficient due to heterogeneity.…”