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  • Title: Time-resolved fluorometry in the diagnosis of Leber hereditary optic neuroretinopathy.
    Author: Huoponen K, Juvonen V, Iitiä A, Dahlen P, Siitari H, Aula P, Nikoskelainen E, Savontaus ML.
    Journal: Hum Mutat; 1994; 3(1):29-36. PubMed ID: 8118464.
    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.
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