A homogeneous europium cryptate-based assay for the diagnosis of mutationsby time-resolved fluorescence resonance energy transfer

Oligonucleotide ligation assay (OLA) is considered to be a very useful meth odology for the detection and characterization of mutations, particularly f or clinical purposes. The fluorescence resonance energy transfer between a fluorescent donor and a suitable fluorophore as acceptor has been applied i n the past to several scientific fields. This technique is well adapted to nucleic acid analysis such as DNA sequencing, DNA hybridization and polymer ase chain reaction. We describe here a homogeneous format based on the use of a rare earth cryptate label as donor: tris-bipyridine-Eu3+. The long-liv ed fluorescence of this label makes it possible to reach a high sensitivity by using a time-resolved detection mode. A non-radiative energy transfer t echnology, known as time-resolved amplification of cryptate emission (TRACE (R)) characterized by a temporal and spectral selectivity has been develope d. The TRACE(R) detection of characterized single nucleotide polymorphism u sing the OLA for allelic discrimination is proposed. We demonstrate the pot entialities of this OLA-TRACE(R) methodology through the analysis of K-ras oncogene point mutations.