Time-resolved detection probe for homogeneous nucleic acid analyses in one-step format

We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay...

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Bibliographic Details
Published in:Analytical biochemistry 2007-02, Vol.361 (1), p.126-131
Main Authors: Laitala, Ville, Ylikoski, Alice, Raussi, Hanna-Mari, Ollikka, Pia, Hemmilä, Ilkka
Format: Article
Language:English
Subjects:
Base Sequence
DNA
DNA - chemistry
DNA - genetics
Energy transfer
Europium
Fluorescence Resonance Energy Transfer - methods
Fluorescent Dyes
FRET
Genetic screening
Homogeneous
Lanthanide
Lanthanoid Series Elements
Multilabel
Multiplex
Nucleic acid
Nucleic Acid Hybridization
Oligodeoxyribonucleotides - chemistry
Polymerase Chain Reaction
Spectrophotometry, Ultraviolet
Time-resolved
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Summary:We report here an extension of homogeneous assays based on fluorescence intensity and lifetime measuring on DNA hybridization. A novel decay probe that allows simple one-step nucleic acid detection with subnanomolar sensitivity, and is suitable for closed-tube applications, is introduced. The decay probe uses fluorescence resonance energy transfer (FRET) between a europium chelate donor and an organic fluorophore acceptor. The substantial change in the acceptor emission decay time on hybridization with the target sequence allows the direct separation of the hybridized and unhybridized probe populations in a time-resolved measurement. No additional sample manipulation or self-hybridization of the probes is required. The wavelength and decay time of a decay probe can be adjusted according to the selection of probe length and acceptor fluorophore, thereby making the probes applicable to multiplexed assays. Here we demonstrate the decay probe principle and decay probe-based, one-step, dual DNA assay using celiac disease-related target oligonucleotides (single-nucleotide polymorphisms [SNPs]) as model analytes. Decay probes showed specific response for their complementary DNA target and allowed good signal deconvolution based on simultaneous optical and temporal filtering. This technique potentially could be used to further increase the number of simultaneously detected DNA targets in a simple one-step homogeneous assay.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2006.11.015