Dolphin Probes: Reagents and Methods for DNA and RNA Detection
- *Abstract
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This invention discloses novel DNA probes that enable detection of high level multiplexing PCR (Polymerase Chain Reaction) or LDR (Ligation Detection Reaction) products and real-time readout. Invented by Cornell researcher Francis Barany and his collaborators, these novel probes are called Dolphin Probes.
As shown in the figures below, Dolphin Probes are comprised of a pair of oligonucleotides bearing donor and acceptor fluorophores that undergo FRET (fluorescence resonance energy transfer) when they are in close proximity to each other. The probes are designed to have predefined melting temperatures with tight melting transitions such that a given probe melts completely over approximately 5 - 10 C. Melting temperatures for Dolphin Probes can be individually tuned through the use of end-capped hairpins and preselected oligonucleotide sequences. The Tm of each Dolphin Probe is a function of the endcap, the stem length and the GC content and order of the nucleotides in the stem. Thus using a single donor-acceptor dye pair, multiple Dolphin Probes may be designed with individually tuned Tm values that are 4-5 C apart. The Dolphin Probes can be used in a multiplexed assay format where the FRET signal is read at different temperatures to generate a melting curve. The melting curve can be de-convoluted to identify the Dolphin Probes that contribute to the FRET signal. A single donor-acceptor FRET pair combined with 10 discretely tuned dolphin probes can allow for 10 individual signals to be detected in a single reaction. Using 6 donor-acceptor dye pairs, up to 60 signals may be multiplexed in a single assay.
When coupled to LDR assays, Dolphin Probes enable multiplexing of between 48- 60 signals. Assays can be performed on standard real time PCR instrumentation such as the ABI 7500 or the Cepheid GeneXpert system, which enables both thermal cycling for the LDR as well as readout of the signals. When used for real time assays analogous to Taqman, dolphin probes allow for multiplexing of up to 18 targets simultaneously.
The invention is unpublished. For earlier, related work by members of this team please see: Wabuyele MB, et al (2003) Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes. J Am Chem Soc.125(23):6937-45.
- *Licensing
- Brian J. Kellybjk44@cornell.edu212-746-6186
- 其他
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None
- 国家/地区
- 美国
