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
H. Hakala et al., 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, NUCL ACID R, 26(24), 1998, pp. 5581-5588
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.