Fluorescent Hybridization Probes Not Requiring Separation Of Products
Description of Invention:
Fluorescent guanosine analogs (excitation at 340 nm, emission at 450 nm) are incorporated into oligonucleotides through a native deoxyribose linkage using automated DNA synthesis which allows them to base stack with native bases. As a result, slight changes in DNA structure can cause significant changes in spectral properties. These compounds are highly fluorescent as monomers in solution, but lose intensity in oligonucleotides. The use of these fluorophores as hairpin hybridization probes is based on the dramatic fluorescence increase that occurs upon them being squeezed out of the strand during annealing where the probe has not been provided with a base-pairing partner in the complementary strand. The degree of increase depends on the oligonucleotide sequence and the annealing strands' concentration. It allows the detection of specific DNA sequences in a mixture without separation of annealed and labeled products. These stable probes are treated as normal phosphoramidites during the DNA synthesis and subsequent de-blocking procedures.
Potential Area of Application:
Molecular probes for PCR-related methodologies in diagnostics of genetic and infectious diseases
Main Advantage of Invention:
Quantum yields of 0.88
50-fold amplification possible upon removal from DNA or bending of DNA
Detection without separation
Stage of Development:
Small-scale synthesis established
Validity demonstrated in various systems, e.g., HIV integrase assay, Alkyl transferase assay
Further Development Required:
Synthesis scale-up to pilot and production levels
Commercial standard quality control and packaging
Low-cost "blue laser" for incorporation into widely used, automated instrumentation techniques
Inventors:
ME Hawkins W Pfleiderer MD Davis FM Balis (NCI)
Patent Status:
DHHS Reference No. E-155-1996/0 --
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Relevant Publication:
* ME Hawkins, W Pfleiderer, A Mazumder, YG Pommier, and FM Balis, "Incorporation of a Fluorescent Guanosine Analog into Oligonucleotides and Its Application to a Real Time Assay for the HIV-1 Integrase 3'-Processing Reaction," Nucleic Acids Research 23 (1995) 2872-2880.
* ME Hawkins, W Pfleiderer, FM Balis, D Porter, JR Knutson, "Fluorescence Properties of Pteridine Nucleoside Analogs as Monomers and Incorporated into Oligonucleotides," Analytical Biochemistry 244 (1997) 86-95.
Licensing Status:
Available for exclusive or non-exclusive licensing
Portfolios: Gene Based Therapies
Gene Based Therapies -Diagnostics Gene Based Therapies -Research Materials
For Additional Information Please Contact: Norbert J. Pontzer PhD JD
NIH Office of Technology Transfer
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Phone: (301) 435-5502
Email: pontzern@mail.nih.gov
Fax: (301) 402-0220
