A homogeneous europium cryptate-based assay for the diagnosis of mutations by time-resolved fluorescence resonance energy transfer.
Lopez-Crapez E, Bazin H, Andre E, Noletti J, Grenier J, Mathis G.
Cancer Research Center, CRLC Val D'Aurelle, Montpellier, France.
Nucleic Acids Res. 2001;29: 14e70.
Oligonucleotide ligation assay (OLA) is considered as a very useful methodology for the detection and characterization of mutations, particularly for clinical purposes. The fluorescence resonance energy transfer (FRET) 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 described here a homogeneous format based on a rare earth cryptate label as donor : tris-bipyridine-Eu3+ [TBP(Eu3+ )]. The long-lived fluorescence of this label allows to reach high sensitivity by using a time-resolved detection mode. A nonradiative energy transfer technology known as Time-Resolved Amplification of Cryptate Emission (TRACE® ) and characterized by a temporal and a spectral selectivity has been developed. The TRACE® detection of characterized single nucleotide polymorphism (SNP) using the OLA for the allelic discrimination is proposed. We demonstrate the potentialities of this OLA-TRACE® methodology through the analysis of K-ras oncogene point mutations.