Time-resolved fluorometry in the diagnosis of Leber hereditary optic neuroretinopathy.
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.
Huoponen
K
K
Department of Medical Genetics, University of Turku, Finland.
Juvonen
V
V
Iitiä
A
A
Dahlen
P
P
Siitari
H
H
Aula
P
P
Nikoskelainen
E
E
Savontaus
M L
ML
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
D004272:DNA, Mitochondrial; D009692:Nucleic Acid Heteroduplexes; D015345:Oligonucleotide Probes; D005063:Europium