title: Time-resolved fluorometry in the diagnosis of Leber hereditary optic neuroretinopathy.
abstract: We have applied time-resolved fluorometry (TRF) to construct a DNA hybridization assay for the diagnosis of Leber hereditary optic neuroretinopathy (LHON). A rapid and reliable detection of the most prevalent mitochondrial DNA (mtDNA) point mutation associated with LHON is demonstrated. In addition, the TRF-method can be used in the quantification of heteroplasmy, a phenomenon commonly present in mtDNA mutations. The assay includes PCR amplification of a fragment encompassing the mutation site followed by hybridization reactions with allele-specific europium (Eu)-labelled oligonucleotide probes. A time-resolved fluorometer is used to measure the bound label. The TRF assay was successfully used to demonstrate the ND4/11778 mutation in patient samples. For quantification of heteroplasmy, synthetic target oligonucleotide mixtures with known ratios of wild-type and mutated sequences were used as standards to control the hybridization step. The assay allowed the detection of heteroplasmy ranging from 5 to 95%. This was also shown in a family with several heteroplasmic members.
authors.affiliation: Department of Medical Genetics, University of Turku, Finland.
authors.forename: K
authors.initials: K
authors.lastname: Huoponen
authors.forename: V
authors.initials: V
authors.lastname: Juvonen
authors.forename: A
authors.initials: A
authors.lastname: Iitiä
authors.forename: P
authors.initials: P
authors.lastname: Dahlen
authors.forename: H
authors.initials: H
authors.lastname: Siitari
authors.forename: P
authors.initials: P
authors.lastname: Aula
authors.forename: E
authors.initials: E
authors.lastname: Nikoskelainen
authors.forename: M L
authors.initials: ML
authors.lastname: Savontaus
mesh_terms: D001483:Base Sequence; D004272:DNA, Mitochondrial / Q000097:blood / Q000235:genetics*; D005063:Europium; D005260:Female; D005470:Fluorometry; D006801:Humans; D007553:Isotope Labeling; D008297:Male; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D015418:Optic Atrophies, Hereditary / Q000175:diagnosis* / Q000235:genetics; D010375:Pedigree; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D004272:DNA, Mitochondrial; D009692:Nucleic Acid Heteroduplexes; D015345:Oligonucleotide Probes; D005063:Europium