Simultaneous detection of several oligonucleotides by time-resolved fluorometry: the use of a mixture of categorized microparticles in a sandwich type mixed-phase hybridization assay

Porous, uniformly sized (50 mu m) glycidyl methacrylate/ethylene dimethacry late particles (SINTEF) were used as a solid phase to construct a sandwich type hybridization assay that allowed simultaneous detection of up to six o ligonucleotides from a single sample, The assay was based on categorization of the particles by two organic prompt fluorophores, viz, fluorescein and dansyl, and quantification of the oligonucleotide hybridization by time-res olved fluorometry. Accordingly, allele-specific oligodeoxyribonucleotide pr obes were assembled on the particles by conventional phosphoramidite strate gy using a non-cleavable linker, and the category defining fluorescein and/ or dansyl tagged building blocks were inserted in the 3'-terminal sequence. An oligonucleotide bearing a photoluminescent europium(III) chelate was hy bridized to the complementary 3'-terminal sequence of the target oligonucle otide, and the resulting duplex was further hybridized to the particle-boun d allele-specific probes via the 5'-terminal sequence of the target. After hybridization each individual particle was subjected to three different flu orescence intensity measurements, The intensity of the prompt fluorescence signals of fluorescein and dansyl defined the particle category, while the europium(III) chelate emission quantified the hybridization. The length of the complementary region between the target oligonucleotide and the particl e-bound probe was optimized to achieve maximal selectivity. Furthermore, th e kinetics of hybridization and the effect of the concentration of the targ et oligomer on the efficiency of hybridization were evaluated, By this appr oach the possible presence of a three base deletion (Delta F508), point mut ation (G542X) and point deletion (1078delT) related to cystic fibrosis coul d unequivocally be detected from a single sample.