GENEALOGY-DNA-L Archives

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Subject: PubMed abstract: detecting mtDNA heteroplasmy
Date: Mon, 1 Aug 2005 19:32:03 EDT

There was some discussion recently about whether all the gazillions of mtDNA
molecules in our body were actually identical. The answer is no, but there
will typically be a dominant variety that is detected by direct sequencing, the
type of testing we have done. The technical word for this mixture is
heteroplasmy. This article is about a technique to detect heteroplasmy at very low
levels of 1%. If I recall correctly, sequencing won't pick up heteroplasmy until
the minor component reaches 20% or so.

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Mitochondrion. 2005 Aug;5(4):282-96.

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

Meierhofer D, Mayr JA, Ebner S, Sperl W, Kofler B.

Department of Paediatrics, Paracelsus Private Medical University Salzburg,
Muellner Hauptstr. 48, A-5020 Salzburg, Austria.

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.

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Ann Turner

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