title: Detection of heteroplasmy in individual mitochondrial particles.
abstract: Mitochondrial DNA (mtDNA) mutations have been associated with disease and aging. Since each cell has thousands of mtDNA copies, clustered into nucleoids of five to ten mtDNA molecules each, determining the effects of a given mtDNA mutation and their connection with disease phenotype is not straightforward. It has been postulated that heteroplasmy (coexistence of mutated and wild-type DNA) follows simple probability rules dictated by the random distribution of mtDNA molecules at the nucleoid level. This model has been used to explain how mutation levels correlate with the onset of disease phenotype and loss of cellular function. Nonetheless, experimental evidence of heteroplasmy at the nucleoid level is scarce. Here, we report a new method to determine heteroplasmy of individual mitochondrial particles containing one or more nucleoids. The method uses capillary cytometry with laser-induced fluorescence detection to detect individual mitochondrial particles stained with PicoGreen, which makes it possible to quantify the mtDNA copy number of each particle. After detection, one or more particles are collected into polymerase chain reaction (PCR) wells and then subjected to real-time multiplexed PCR amplification. This PCR strategy is suitable to obtain the relative abundance of mutated and wild-type mtDNA. The results obtained here indicate that individual mitochondrial particles and nucleoids contained within these particles are not heteroplasmic. The results presented here suggest that current models of mtDNA segregation and distribution (i.e., heteroplasmic nucleoids) need further consideration.
authors.affiliation: Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
authors.forename: Bobby G
authors.initials: BG
authors.lastname: Poe
authors.forename: Ciarán F
authors.initials: CF
authors.lastname: Duffy
authors.forename: Michael A
authors.initials: MA
authors.lastname: Greminger
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Nelson
authors.forename: Edgar A
authors.initials: EA
authors.lastname: Arriaga
mesh_terms: D002460:Cell Line; D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics*; D018628:Gene Dosage; D005838:Genotype; D006801:Humans; D047569:Laser Scanning Cytometry / Q000379:methods; D009154:Mutation; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D004272:DNA, Mitochondrial
references.citation: Anal Bioanal Chem. 2006 Jan;384(1):169-74
references.pmid: 16320040
references.citation: Int J Biochem Cell Biol. 2009 Oct;41(10):1899-906
references.pmid: 19703654
references.citation: Eur J Biochem. 1998 Oct 1;257(1):192-201
references.pmid: 9799119
references.citation: Mol Biol Cell. 1999 Oct;10(10):3345-56
references.pmid: 10512871
references.citation: Exp Cell Res. 1991 Sep;196(1):137-40
references.pmid: 1715276
references.citation: Science. 1989 Oct 27;246(4929):500-3
references.pmid: 2814477
references.citation: J Neurol. 2003 Mar;250(3):267-77
references.pmid: 12638015
references.citation: J Chromatogr A. 2006 Dec 29;1137(2):249-55
references.pmid: 17070532
references.citation: Hum Mol Genet. 1996 Feb;5(2):197-205
references.pmid: 8824875
references.citation: Bioessays. 2000 Jun;22(6):564-72
references.pmid: 10842310
references.citation: Anal Bioanal Chem. 2007 Jan;387(1):107-18
references.pmid: 16937092
references.citation: Science. 2005 Jun 24;308(5730):1909-11
references.pmid: 15879174
references.citation: Anal Chem. 2004 Jan 15;76(2):315-21
references.pmid: 14719877
references.citation: Mol Biol Cell. 2003 Apr;14(4):1583-96
references.pmid: 12686611
references.citation: Nature. 2010 Mar 25;464(7288):610-4
references.pmid: 20200521
references.citation: Anal Chem. 2007 Oct 15;79(20):7691-9
references.pmid: 17877423
references.citation: Anal Biochem. 2007 Mar 15;362(2):193-200
references.pmid: 17270140
references.citation: Exp Cell Res. 2000 Aug 25;259(1):79-85
references.pmid: 10942580
references.citation: Biochim Biophys Acta. 2010 Mar;1800(3):245-9
references.pmid: 19616602
references.citation: Muscle Nerve. 2006 Sep;34(3):265-83
references.pmid: 16810684
references.citation: Electrophoresis. 1998 Jul;19(10):1677-82
references.pmid: 9719545
references.citation: Science. 2005 Jul 15;309(5733):481-4
references.pmid: 16020738
references.citation: Nature. 2004 May 27;429(6990):417-23
references.pmid: 15164064
references.citation: Anal Biochem. 1997 Jul 1;249(2):228-38
references.pmid: 9212875
references.citation: Exp Cell Res. 2005 Feb 15;303(2):432-46
references.pmid: 15652355
references.citation: Oncogene. 2006 Aug 7;25(34):4663-74
references.pmid: 16892080
references.citation: Eur J Pediatr. 2000 Dec;159 Suppl 3:S219-26
references.pmid: 11216904