E. Lopez-crapez et al., A homogeneous europium cryptate-based assay for the diagnosis of mutationsby time-resolved fluorescence resonance energy transfer, NUCL ACID R, 29(14), 2001, pp. NIL_4
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.