Intergen Company

Serologicals, Inc.

Glenn Nardone

Irena Nazarenko

Jila Boal

An oligonucleotide, labeled with a molecular energy transfer trio and containing two sequences capable of hairpin formation, is used in the detection of two targets by irradiation with a single wavelength of light. One of the two sequences contains an energy donor and a first energy acceptor, and the other sequence contains a second energy acceptor. The donor is in close proximity to the second acceptor only if the hairpin is formed, while the donor is always in close proximity to first acceptor. A sample is assayed, using this oligonucleotide in conjunction with another oligonucleotide which contains the donor fluorophore and the quencher, arranged as described above, but which lacks the acceptor fluorophore. The present oligonucleotide and the other oligonucleotide are specific to first and second targets, respectively. If a sample contains the first and second targets, then hairpin formation is prevented after each oligonucleotide is incorporated into a target amplification product or hybridized to a target. Subsequent irradiation of the sample with the single wavelength of light which excites the donor fluorophore, but not the acceptor fluorophore, causes two distinctive signals to be generated. The first signal is emitted by the second fluorophore of the present oligonucleotide, while the second signal is emitted by the first fluorophore of the other oligonucleotide. Thus, the first and second targets are detected when one observes the first and second signals, respectively.

Multi-fluorescent hairpin energy transfer oligonucleotides

Compounds are described comprising purines substituted at the C-8 position and pyrimidines substituted at the C-4 position with a linker and quencher. These compounds, when incorporated into hairpin oligonucleotides quench the fluorescence of fluorophores linked to the 5&prime;-terminus of the oligonucleotide. These nucleotide-quencher compounds are also easily incorporated into oligonucleotides using conventional or automated oligonucleotide synthetic techniques.

Purines and pyrimidines linked to a quencher

Compounds are described comprising pyrimidines substituted at the C-5 position with a linker and quencher. These compounds when incorporated into hairpin oligonucleotides quench the fluorescence of fluorophores linked to the 5'-terminus of the oligonucleotide. These nucleotide-quencher compounds are also easy to incorporate into oligonucleotides using conventional, automated, oligonucleotide synthetic techniques.

Growing community of inventors

Oakton, VA, United States of America

Glenn Nardone