Physical Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, United States of America.
PLoS One. 2010 Dec 16;5(12):e14359. doi: 10.1371/journal.pone.0014359.
Mitochondrial DNA (mtDNA) genome mutations can lead to energy and respiratory-related disorders like myoclonic epilepsy with ragged red fiber disease (MERRF), mitochondrial myopathy, encephalopathy, lactic acidosis and stroke (MELAS) syndrome, and Leber's hereditary optic neuropathy (LHON). It is not well understood what effect the distribution of mutated mtDNA throughout the mitochondrial matrix has on the development of mitochondrial-based disorders. Insight into this complex sub-cellular heterogeneity may further our understanding of the development of mitochondria-related diseases.
This work describes a method for isolating individual mitochondria from single cells and performing molecular analysis on that single mitochondrion's DNA. An optical tweezer extracts a single mitochondrion from a lysed human HL-60 cell. Then a micron-sized femtopipette tip captures the mitochondrion for subsequent analysis. Multiple rounds of conventional DNA amplification and standard sequencing methods enable the detection of a heteroplasmic mixture in the mtDNA from a single mitochondrion.
Molecular analysis of mtDNA from the individually extracted mitochondrion demonstrates that a heteroplasmy is present in single mitochondria at various ratios consistent with the 50/50 heteroplasmy ratio found in single cells that contain multiple mitochondria.
线粒体 DNA（mtDNA）基因组突变可导致能量和呼吸相关的疾病，如肌阵挛性癫痫伴破碎红纤维病（MERRF）、线粒体肌病、脑病、乳酸酸中毒和卒中（MELAS）综合征和莱伯遗传性视神经病变（LHON）。mtDNA 突变在整个线粒体基质中的分布如何影响线粒体疾病的发展，目前尚不清楚。深入了解这种复杂的亚细胞异质性可能有助于我们理解线粒体相关疾病的发生。
本工作描述了一种从单个细胞中分离单个线粒体并对该单个线粒体的 DNA 进行分子分析的方法。光镊从裂解的人 HL-60 细胞中提取单个线粒体。然后，微米级的 femtopipette 尖端捕获线粒体，以备后续分析。多次常规 DNA 扩增和标准测序方法可检测单个线粒体 mtDNA 中的异质混合物。
从单个提取的线粒体进行 mtDNA 的分子分析表明，在不同比例下存在异质性，与含有多个线粒体的单个细胞中发现的 50/50 异质性比例一致。
