domingo, 16 de enero de 2011
PLoS ONE: Detection of Heteroplasmic Mitochondrial DNA in Single Mitochondria
Detection of Heteroplasmic Mitochondrial DNA in Single Mitochondria
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.
Joseph E. Reiner1*, Rani B. Kishore1, Barbara C. Levin2, Thomas Albanetti3, Nicholas Boire3, Ashley Knipe3, Kristian Helmerson1, Koren Holland Deckman3
1 Physical Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, United States of America, 2 Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, United States of America, 3 Department of Chemistry, Gettysburg College, Gettysburg, Pennsylvania, United States of America
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.
Citation: Reiner JE, Kishore RB, Levin BC, Albanetti T, Boire N, et al. (2010) Detection of Heteroplasmic Mitochondrial DNA in Single Mitochondria. PLoS ONE 5(12): e14359. doi:10.1371/journal.pone.0014359
Editor: Tadafumi Kato, RIKEN Brain Science Institution, Japan
Received: March 23, 2010; Accepted: October 27, 2010; Published: December 16, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: The authors have no support or funding to report.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: joseph.reiner@nist.gov
full-text (large):
PLoS ONE: Detection of Heteroplasmic Mitochondrial DNA in Single Mitochondria
Suscribirse a:
Enviar comentarios (Atom)
No hay comentarios:
Publicar un comentario