Next generation sequencing to characterize mitochondrial genomic DNA heteroplasmy.
Next generation sequencing to characterize mitochondrial genomic DNA heteroplasmy.
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DOI：
10.1002/0471142905.hg1908s71
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发表时间：
2011-10-01
影响因子：
--
通讯作者：
Huang, Taosheng
中科院分区：
文献类型：
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作者：
Huang, Taosheng
本方案描述了通过平行测序鉴定线粒体DNA(MtDNA)异质性的方法学。线粒体在细胞的基本功能中起着重要的作用。每个真核细胞包含数百个线粒体和数百个线粒体基因组。突变型和野生型线粒体DNA可能以异质性共存，并导致人类疾病。该方法的目的是同时测定线粒体DNA序列，并对异质性水平进行量化。该方法包括两个片段的线粒体基因组DNA聚合酶链式反应扩增。然后，将PCR产物以等摩尔比混合。然后用高通量的下一代测序技术对样本进行条形码和测序。这项技术在确定线粒体DNA突变和异质性水平方面具有高度的敏感性、特异性和准确性。
This protocol describes the methodology to characterize mitochondrial DNA (mtDNA) heteroplasmy by parallel sequencing. Mitochondria play an important role in essential cellular functions. Each eukaryotic cell contains hundreds of mitochondria with hundreds of mitochondria genomes. Mutant and wild-type mtDNA may co-exist as heteroplasmy, and cause human disease. The purpose of this protocol is to simultaneously determine mtDNA sequence and quantify the heteroplasmic level. This protocol includes a two-fragment mitochondrial genome DNA PCR amplification. The PCR product is then mixed at an equimolar ratio. 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 level of heteroplasmy.