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|Author(s):||Joseph E. Reiner; Rani Kishore; Barbara C. Levin; Thomas Albanetti; Nicholas Boire; Ashley Knipe; Kristian Helmerson; Koren Deckman;|
|Title:||Detection of Heteroplasmic Mitochondrial DNA in Single Mitochondria|
|Published:||December 17, 2010|
|Abstract:||Background 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. Methodology 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. Significance 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.|
|Keywords:||nanobiotechnology, optical tweezers, mitochondria, DNA technologies|
|Research Areas:||Life Sciences Research|
|PDF version:||Click here to retrieve PDF version of paper (451KB)|