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

• 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
• ISSN: 0022-1554; 1551-5044; 1551-5044
• DOI: 10.1369/jhc.7A7282.2007

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.