Department of Pediatrics, Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, New York.
Curr Protoc. 2022 May;2(5):e412. doi: 10.1002/cpz1.412.
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 high-throughput next-generation sequencing technology. This technology is highly sensitive, specific, and accurate in determining mtDNA mutations and the degree/level of heteroplasmy. © 2022 Wiley Periodicals LLC. Basic Protocol 1: PCR amplification of mitochondrial DNA Basic Protocol 2: Analysis of next-generation sequencing of mitochondrial DNA Basic Protocol 3: Mutect2 pipeline for automated sample processing and large-scale data analysis.
线粒体在许多关键的细胞功能中起着非常重要的作用。每个真核细胞都含有数百个线粒体，每个线粒体都有数百个线粒体基因组。突变型和野生型线粒体 DNA（mtDNA）可能以异质性的形式共存，并导致人类疾病。本文中方案的目的是使用平行测序同时确定 mtDNA 序列和量化异质性水平。该方案包括使用两组不同的 PCR 引物对两个全长产物进行线粒体基因组 DNA PCR 扩增。将 PCR 产物按等摩尔比混合，然后对样品进行条形码标记，并使用高通量下一代测序技术进行测序。该技术在确定 mtDNA 突变和异质性程度/水平方面具有高度的敏感性、特异性和准确性。© 2022 Wiley Periodicals LLC. 基本方案 1：线粒体 DNA 的 PCR 扩增 基本方案 2：线粒体 DNA 下一代测序分析 基本方案 3：Mutect2 流水线用于自动化样本处理和大规模数据分析。
