Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-01-17
2004-07-27
Fredman, Jeffrey (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024310, C536S024330, C435S006120
Reexamination Certificate
active
06768000
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is broadly concerned with an oligonucleotide probe useful for detecting multiple target nucleic acid sequences in a sample. More particularly, the present invention relates to an oligonucleotide probe labeled with a molecular energy transfer (MET) trio including an energy donor and two energy acceptors which makes possible the detection of the multiple targets using only one excitation wavelength of light.
2. Description of the Related Art
The MET phenomenon is a process by which energy is passed between a donor molecule and an acceptor molecule. Fluorescence resonance energy transfer (FRET), which involves at least one fluorophore, is a form of MET. A fluorophore is a compound that absorbs light at one wavelength, and emits light at different wavelength. A spectrofluorimeter is used to simultaneously emit light which excites the fluorophore, and detect light emitted by the fluorophore. In FRET, the fluorophore is a donor molecule which absorbs photons, and subsequently transfers this energy to an acceptor molecule. Donor and acceptor molecules that engage in MET or FRET are termed MET pairs or FRET pairs, respectively. Förster, 1949, Z. Naturforsch A4:321-327; Clegg, 1992, Methods In Enzymology 211:353-388.
When two fluorphores are in close proximity, and the emission spectrum of the first fluorophore overlaps the excitation spectrum of the second fluorophore, excitation of the first fluorophore causes it to emit light that is absorbed by the second fluorophore, which in turn causes the second fluorophore to emit light. As a result, the fluorescence of the first fluorophore is quenched, while the fluorescence of the second flourophore is enhanced. If the energy of the first fluorophore is transferred to a compound that is not a fluorophore, however, the fluorescence of the first fluorophore is quenched without subsequent emission of light by the non-fluorophore.
The FRET phenomenon has been exploited in methods for detecting nucleic acids. One of these methods is disclosed in U.S. Pat. No. 5,866,366, the entire contents of which are herein incorporated by reference. The '366 patent discloses a FRET-labeled hairpin oligonucleotide which is used as a probe in polymerase chain reaction (PCR) methods to detect target nucleic acid sequences. This oligonucleotide contains an energy donor and an energy acceptor constituting a FRET pair. The donor and acceptor are respectively situated on first and second nucleotide sequences of the oligonucleotide. These two nucleotide sequences are complementary to each other, and are therefore able to form a hairpin in the oligonucleotide.
If the first and second nucleotide sequences are annealed to each other, then the donor and acceptor are in close proximity. In this spatial arrangement, the acceptor absorbs the emission from the donor, and thereby quenches the signal from the donor. However, if the nucleotide sequences are not annealed to each other, then the donor and acceptor are separated, the acceptor can no longer absorb the emission from the donor, and the signal from the donor is not quenched.
Thus, if the oligonucleotide is incorporated into an amplification product during PCR, then the hairpin unfolds, resulting in the separation of the donor from the acceptor, and the consequent emission of an observable signal. However, if the oligonucleotide is not incorporated into a PCR amplification product, then the hairpin remains, and the emission from the donor is quenched by the acceptor. Detection of a signal after PCR therefore indicates the presence of the target.
Additionally, the FRET-labeled hairpin oligonucleotide described above may be used as a “molecular beacon” to detect a target nucleic acid sequence without incorporating it into a DNA molecule. The molecular-beacon technology is described in Tyagi et al., 1996, Nature Biotechnology 14:303-308, the entire contents of which are herein incorporated by reference. If the oligonucleotide hybridizes to a target, the hairpin unfolds, and a detectable signal is generated. If the oligonucleotide does not hybridize to the target, the hairpin remains, and the signal is quenched. Detection of a signal after hybridization therefore indicates the presence of the target.
Detection of more than one target nucleic acid molecule in a single sample using the methods disclosed in the '336 patent and Tyagi et al. requires a FRET-labeled hairpin oligonucleotide specific to each target molecule, wherein each donor emits a distinctive signal. Since each donor typically must be excited by a different wavelength of light, it is necessary to irradiate the sample with multiple wavelengths of light. A major drawback to this approach, however, is that it requires a spectrofluorimeter emitting a broad spectrum of light.
SUMMARY OF THE INVENTION
Avoiding the aforementioned drawback, the present invention is directed to a MET-labeled oligonucleotide, as well as to the use of this oligonucleotide to detect multiple target nucleic acid sequences in a single sample. Pursuant to this invention, multiple targets can be detected by irradiating a sample, containing the targets, with a single excitation wavelength of light.
An oligonucleotide according to the present invention contains three nucleotide sequences: a first nucleotide sequence, a second nucleotide sequence at the 5′ end of the first nucleotide sequence, and a third nucleotide sequence at the 5′ end of the second nucleotide sequence. Additionally, the oligonucleotide contains a MET trio, e.g a FRET trio, that includes an energy donor moiety and first and second energy acceptor moieties, where (i) the energy donor moiety is capable of emitting a quantum of energy and (ii) each of the first and second acceptor moieties is capable of absorbing a substantial amount of the quantum of energy. Preferably, the first acceptor moiety also is capable of emitting a quantum of energy.
The donor moiety is attached to a nucleotide of the first nucleotide sequence, the first acceptor moiety is attached to a nucleotide of the first nucleotide sequence, and the second acceptor moiety is attached to a nucleotide of the third nucleotide sequence, or, alternatively, the donor moiety is attached to a nucleotide of the third nucleotide sequence, the first acceptor moiety is attached to a nucleotide of the third nucleotide sequence, and the second acceptor moiety is attached to a nucleotide of the first nucleotide sequence.
The oligonucleotide is capable of forming a hairpin containing a nucleotide of the first nucleotide sequence and a nucleotide of the third nucleotide sequence. If the donor moiety emits the quantum of energy, then the first acceptor moiety absorbs a substantial amount of the emitted quantum of energy if, preferably only if, the hairpin is not formed, and the second acceptor moiety absorbs a substantial amount of the emitted quantum of energy if, preferably only if, the hairpin is formed.
An oligonucleotide of this invention preferably comprises a fourth nucleotide sequence at the 3′ end of the first nucleotide sequence. Ideally, the third nucleotide sequence is not complementary to the fourth nucleotide sequence, and the fourth nucleotide sequence is complementary to a nucleotide sequence flanking a target nucleotide sequence. e.g., a DNA sequence. The oligonucleotide may be included in a kit containing a polymerase. The oligonucleotide preferably comprises a deoxyribonucleotide.
Advantageously, the donor moiety and the first acceptor moiety each are fluorophores, while the second acceptor moiety is a quencher of light emitted by the donor moiety. The preferred donor moiety, first acceptor moiety, and second acceptor moiety are fluorescein, 6-carboxy-X-rhodamine (ROX), and 4-(4′-dimethylamino-phenylazo) benzoic acid (DABSYL), respectively.
Preferably, there is (are) 0 to 50 nucleotide(s) in between the nucleotide to which the donor moiety is attached and the nucleotide to which the first acceptor moiety is attached, and there is (are) 0 to 50 nucleoti
Chunduru Suryaprabha
Fredman Jeffrey
Intergen Company
LandOfFree
Multi-fluorescent hairpin energy transfer oligonucleotides does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Multi-fluorescent hairpin energy transfer oligonucleotides, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Multi-fluorescent hairpin energy transfer oligonucleotides will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3241020