An exciplex-based, target-assembled fluorescence system with inherently low background to probe for specific nucleic acid sequences.
A novel detection technique, called ExciProbes, has been developed to proof-of-principle level for DNA oligonucleotides. The new approach is based on the use of two short oligonucleotides complementary to a target nucleic acid sequence. Each short-probe oligonucleotide bears the separated parts of a new class of fluorescence detector, an exciplex. These isolated parts of the detector have no inherent signal at the detection wavelength. They are designed to detect biotarget by being assembled by the target itself to give a new molecular entity (the exciplex), with a characteristic fluorescence and very large Stokes shift (typically >150 nm). The technique is not related to fluorescence resonance energy transfer, and can potentially resolve to 1 base pair. ExciProbes can detect single or double mutations in a short sequence of DNA, and can be combined with temperature-filtering to provide allelic discrimination of single nucleotide polymorphism analysis. Compared to other fluorophore systems that have large backgrounds (typically >60%), ExciProbes show backgrounds of <1% under comparable conditions, and can be used with DNA, RNA, or synthetic nucleic acids such as locked nucleic acid.
Bichenkova
Elena V
EV
Wolfson Centre for Structure-Based Rational Design of Molecular Diagnostics, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester, UK. Elena.Bichenkova@manchester.ac.uk
Sardarian
Ali
A
Savage
Hannah E
HE
Rogert
Candelaria
C
Douglas
Kenneth T
KT
D004252:DNA Mutational Analysis / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D015345:Oligonucleotide Probes