Abstract
Mitochondrial DNA (mtDNA) mutations are commonly found in the skeletal muscle of patients with mitochondrial disease, inflammatory myopathies and sarcopenia. The majority of these mutations are mtDNA deletions, which accumulate to high levels in individual muscle fibres causing a respiratory defect. Most mtDNA deletions are major arc deletions with breakpoints located between the origin of light strand (OL) and heavy strand (OH) replication within the major arc. However, under certain disease conditions, rarer, minor arc deletions are detected. Currently, there are few techniques which would allow the detection and quantification of both types of mtDNA deletions in single muscle fibres. We have designed a novel triplex real-time PCR assay which simultaneously amplifies the MT-ND4 gene in the major arc, the MT-ND1 gene in the minor arc, and the non-coding D-Loop region. We demonstrate that this assay is a highly sensitive and reliable tool for the detection and quantification of a broad range of major and minor arc mtDNA deletions with the potential to investigate the molecular pathogenesis in both research and diagnostic settings.
Highlights
The gold standard technique for the detection and quantitative assessment of mtDNA deletions is Southern blotting; this requires large quantities of DNA and is not suitable for small samples such as single cell preparations[16]
An alternative method, developed in our laboratory, uses TaqMan real-time PCR chemistry to perform a simultaneous analysis of two mitochondrial genes; MT-ND1 that is rarely deleted and MT-ND4 which is often deleted as the majority of deletions appear within the major arc[18,19,20]
Our duplex real-time PCR assay is a useful tool for quantifying major arc deletions of the mitochondrial genome, it fails to detect and quantify rarer mtDNA deletions which extend into the minor arc deleting or partially deleting both MT-ND1 and MT-ND4
Summary
The gold standard technique for the detection and quantitative assessment of mtDNA deletions is Southern blotting; this requires large quantities of DNA and is not suitable for small samples such as single cell preparations[16]. In both ageing and a variety of disease pathologies, mtDNA deletions accumulate to high levels in individual cells, but affect only a minor proportion of mtDNA molecules in tissue homogenate. A three primer real-time PCR assay has been historically used to quantify the level of a specific mtDNA deletion in transmitochondrial cybrid cells[17] This method relies on a pair of probes positioned between a forward and one of two reverse primers. We show that this triplex real-time PCR assay can accurately quantify mtDNA deletion load in single cells from both ageing and disease tissues
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