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.

Figure 1

OLA with TRACE^® detection. A symmetric PCR encompassing the bases to analyze was made. In the semi-direct format a common probe: 5′-phosphorylated (p) and 3′-[TBP(Eu³⁺)]-labeled and an allele-specific probe: 5′-biotinylated (bio) containing at its 3′-end the specific base, were reacted with the PCR products in a cycling mode. Perfect hybrids (allele-specific probe: PCR product) allow the generation of a ligation product 5′-biotinylated and 3′-[TBP(Eu³⁺)]-labeled. After the addition of the streptavidin conjugate (SA-XL665 or SA-Cy5), fluorescence energy transfer was monitored on a dual wavelength fluorimeter. In the direct format, the allele-specific probe was 5′-Cy5-labeled, then after the cycles of hybridization/ligation, the products were directly analyzed.

Figure 2

Chemical structure of [TBP(Eu³⁺)]-labeled common probes.

Figure 3

Temperature optimization of the OLA–TRACE^®. OLA–TRACE^® reactions were performed as described in Materials and Methods at various cycling temperatures by using 800 fmol of common [TBP(Eu³⁺)]-labeled probe (KRCPA1–15), 800 fmol of allele-specific probe (RWB1–15, wild-type GGT K-ras codon 12 sequence), 800 fmol of SA-XL665, and an aliquot of the PCR amplified cell line to analyze LnCaP (GGT, circles) or Mia PaCa-2 (TGT, diamonds).

Figure 4

Specificity of the OLA–TRACE^® technology by analyzing synthetic DNAs. Seven synthetic DNAs containing the seven K-ras codon 12 (GGT, GAT, GCT, GTT, GAT, GCT and GTT) possible sequences were analyzed at the same time by eight pairs of 20-base-long OLA probes in the semi-direct format. The results represent the mean of two consecutive assays. The relative Delta F value 100 was assigned to the reaction between full complementary sequences.

Figure 5

Sensitivity of detection of K-ras mutant alleles. DNA from Mia PaCa-2 (TGT) and LnCaP (GGT) cell lines were diluted and the OLA products which were generated by using the allele-specific TGT probe RM3B-20 (white columns) or the allele-specific GGT probe RWB1-20 (black columns), were analysed by TRACE^®.