title: Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations.
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.
authors.affiliation: Department of Paediatrics, Paracelsus Private Medical University Salzburg, Muellner Hauptstr. 48, A-5020 Salzburg, Austria.
authors.forename: David
authors.initials: D
authors.lastname: Meierhofer
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Mayr
authors.forename: Sabine
authors.initials: S
authors.lastname: Ebner
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sperl
authors.forename: Barbara
authors.initials: B
authors.lastname: Kofler
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics*; D006801:Humans; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D004272:DNA, Mitochondrial