Single Cell A3243G Mitochondrial DNA Mutation Load Assays for Segregation Analysis

Roshan S. Jahangir Tafrechi ¹, Frans M. van de Rijke ¹, Amin Allallou ¹, Chatarina Larsson ¹, Willem C.R. Sloos ¹, Marchien van de Sande ¹, Carolina Wählby ¹, George M.C. Janssen ¹ and Anton K. Raap ^1*

¹ 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

^* To whom correspondence should be addressed. E-mail: a.k.raap{at}lumc.nl.

Submitted on May 1, 2007
Accepted on 13 July 2007

	Abstract

Segregation of 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 base 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-RFLP 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.

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

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