title: A homogeneous europium cryptate-based assay for the diagnosis of mutations by time-resolved fluorescence resonance energy transfer.
abstract: Oligonucleotide ligation assay (OLA) is considered to be a very useful methodology for the detection and characterization of mutations, particularly for clinical purposes. The fluorescence resonance energy transfer between a fluorescent donor and a suitable fluorophore as acceptor has been applied in the past to several scientific fields. This technique is well adapted to nucleic acid analysis such as DNA sequencing, DNA hybridization and polymerase chain reaction. We describe here a homogeneous format based on the use of a rare earth cryptate label as donor: tris-bipyridine-Eu(3+). The long-lived fluorescence of this label makes it possible to reach a high sensitivity by using a time-resolved detection mode. A non-radiative energy transfer technology, known as time-resolved amplification of cryptate emission (TRACE((R))) characterized by a temporal and spectral selectivity has been developed. The TRACE((R)) detection of characterized single nucleotide polymorphism using the OLA for allelic discrimination is proposed. We demonstrate the potentialities of this OLA-TRACE((R)) methodology through the analysis of K-ras oncogene point mutations.
authors.affiliation: Centre de Recherche en Cancérologie, C.R.L.C. Val d'Aurelle, Parc Euromédecine, 34298 Montpellier Cedex 5, France. ecrapez@valdorel.fnclcc.fr
authors.forename: E
authors.initials: E
authors.lastname: Lopez-Crapez
authors.forename: H
authors.initials: H
authors.lastname: Bazin
authors.forename: E
authors.initials: E
authors.lastname: Andre
authors.forename: J
authors.initials: J
authors.lastname: Noletti
authors.forename: J
authors.initials: J
authors.lastname: Grenier
authors.forename: G
authors.initials: G
authors.lastname: Mathis
mesh_terms: D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D011905:Genes, ras / Q000235:genetics; D006801:Humans; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D009942:Organometallic Compounds / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014407:Tumor Cells, Cultured
chemical_list: D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D009942:Organometallic Compounds; C070734:europium(III) trisbipyridine cryptate