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Originally published as JHC exPRESS on August 6, 2007.
doi:10.1369/jhc.7A7282.2007
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Journal of Histochemistry and Cytochemistry
Volume 55 (11): 1159-1166, 2007
Copyright ©The Histochemical Society, Inc.

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

Roshan S. Jahangir Tafrechi, Frans M. van de Rijke, Amin Allallou, Chatarina Larsson, Willem C.R. Sloos, Marchien van de Sande, Carolina Wählby, George M.C. Janssen and Anton K. Raap

Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands (RSJT,FMvdR,WCRS,MvdS,GMCJ,AKR), and Department of Genetics and Pathology (CL,CW) and Center for Image Analysis (AA,CW), Uppsala University, Uppsala, Sweden

Correspondence to: Anton K. Raap, Department of Molecular Cell Biology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands. E-mail: a.k.raap{at}lumc.nl

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. (J Histochem Cytochem 55:1159–1166, 2007)

Key Words: segregation • heteroplasmy • mitochondrial diseases • aging • A3243G mtDNA • mutation load • Padlock probing • PCR-RFLP


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