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 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.
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
Roshan S
RS
Jahangir Tafrechi
Frans M
FM
van de Rijke
Amin
A
Allallou
Chatarina
C
Larsson
Willem C R
WC
Sloos
Marchien
M
van de Sande
Carolina
C
Wählby
George M C
GM
Janssen
Anton K
AK
Raap
D002469:Cell Separation; D002478:Cells, Cultured; D004272:DNA, Mitochondrial / Q000235:genetics*; D005838:Genotype; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D044366:Transition Temperature
D004272:DNA, Mitochondrial