Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic 
mutations

Meierhofer, David; Mayr, Johannes A.; Ebner, Sabine; Sperl, Wolfgang; Kofler, Barbara

doi:10.1016/j.mito.2005.06.001

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Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations

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Meierhofer, David
Mass Spectrometry (Head: David Meierhofer), Scientific Service (Head: Christoph Krukenkamp), Max Planck Institute for Molecular Genetics, Max Planck Society;
Department of Paediatrics, Paracelsus Private Medical University Salzburg, A-5020 Salzburg, Austria;

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Citation

Meierhofer, D., Mayr, J. A., Ebner, S., Sperl, W., & Kofler, B. (2005). Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations. Mitochondrion, 5(4), 282-296. doi:10.1016/j.mito.2005.06.001.

Cite as: https://hdl.handle.net/11858/00-001M-0000-0028-8F55-9

Abstract

Alterations of the mitochondrial DNA (mtDNA) are implicated in various pathological conditions. In this study, we used denaturing high performance liquid chromatography (DHPLC) as a method to rapidly screen the entire mtDNA for mutations. Overlapping DNA fragments, amplified by one single cycling protocol from frozen pre-formulated PCR mixes, were subjected to DHPLC analysis. Single DHPLC injections of fragments yielded straightforward interpretation of results with a detection limit down to 1% mtDNA heteroplasmy. Furthermore, collection and re-amplification of low degree heteroduplex peak-fractions allowed sequence analysis of mtDNA mutations down to the detection limit of the DHPLC method. In order to demonstrate that the method has diagnostic value, we analyzed and confirmed known mtDNA mutations in patient samples.