Single-cell A3243G Mitochondrial DNA Mutation Load Assays for Segregation Analysis

Single-cell A3243G Mitochondrial DNA Mutation Load Assays for Segregation Analysis

Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the pa...

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Bibliographic Details
Published in:The journal of histochemistry and cytochemistry Vol. 55; no. 11; pp. 1159 - 1166
Main Authors: Jahangir Tafrechi, Roshan S, van de Rijke, Frans M, Allallou, Amin, Larsson, Chatarina, Sloos, Willem C.R, van de Sande, Marchien, Wahlby, Carolina, Janssen, George M.C, Raap, Anton K
Format: Journal Article
Language:English
Published: Los Angeles, CA Histochemical Soc 01-11-2007
SAGE Publications
Subjects:
A3243G mtDNA
Aging
Cell Separation
Cells, Cultured
DNA, Mitochondrial - genetics
Genotype
Heteroplasmy
Humans
Image Processing, Computer-Assisted
MEDICIN
MEDICINE
Microscopy, Fluorescence
Mitochondrial diseases
Mutation
Mutation load
Padlock probing
PCR-RFLP
Polymerase Chain Reaction - methods
Polymorphism, Restriction Fragment Length
Segregation
Transition Temperature
heteroplasmy
mitochondrial diseases
mutation load
A3243G mtDNA
aging
PCR-RFLP
segregation
Padlock probing
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Summary:Segregation of mitochondrial DNA (mtDNA) is an important underlying pathogenic factor in mtDNA mutation accumulation in mitochondrial diseases and aging, but the molecular mechanisms of mtDNA segregation are elusive. Lack of high-throughput single-cell mutation load assays lies at the root of the paucity of studies in which, at the single-cell level, mitotic mtDNA segregation patterns have been analyzed. Here we describe development of a novel fluorescence-based, non-gel PCR restriction fragment length polymorphism method for single-cell A3243G mtDNA mutation load measurement. Results correlated very well with a quantitative in situ Padlock/rolling circle amplification—based genotyping method. In view of the throughput and accuracy of both methods for single-cell A3243G mtDNA mutation load determination, we conclude that they are well suited for segregation analysis.
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ISSN:0022-1554
1551-5044
1551-5044
DOI:10.1369/jhc.7A7282.2007