Duplex stabilizing fluorescence quenchers for nucleic acid probes
Diaryl-azo derivatives are efficient fluorescence quenchers as well as nucleic acid duplex-stabilizing agents and are useful in oligonucleotide conjugates and probes. The oligonucleotide-quencher conjugates may be used in detection methods for nucleic acid targets.
1. A method for detecting a target nucleic acid sequence in a sample, comprising:
contacting the sample with an oligonucleotide conjugate comprising a fluorescence quenching compound and having the formula:
wherein ODN is an oligonucleotide,
L is a linking group having from 0 to 100 main chain atoms selected from C, N, O, S, P and Si, wherein L can include acyclic, cyclic or aromatic groups, or combinations thereof,
R 1 is H, alkyl, or alkyl covalently connected to Ar 3 ,
Ar 1 and Ar 2 are aromatic or hetero-aromatic moieties,
Ar 3 is an aromatic moiety,
R 3 is hydroxyl, a linking group, or a blocking group, and
n is from 1 to 5,
wherein the oligonucleotide conjugate has a nucleic acid sequence at least partially complementary to the target nucleic acid sequence, and wherein the oligonucleotide conjugate further comprises a fluorophore; and
detecting a fluorescent signal resulting from hybridization of the oligonucleotide conjugate to the target nucleic acid sequence.
2. The method of claim 1 , wherein the fluorescent signal is generated by action of a polymerase having 5′-nuclease activity.
3. The method of claim 1 , wherein the oligonucleotide conjugate is a primer or a probe.
4. The method of claim 1 , further comprising the step of amplifying the target nucleic acid sequence.
5. A method for detecting a target nucleic acid sequence in a sample, comprising:
contacting the sample with an oligonucleotide conjugate having the formula:
wherein n is 1, 2, or 3,
wherein the oligonucleotide conjugate has a nucleic acid sequence at least partially complementary to the target nucleic acid sequence, and wherein the oligonucleotide conjugate further comprises a fluorophore; and
detecting a fluorescent signal resulting from hybridization of the oligonucleotide conjugate to the target nucleic acid sequence.
6. The method of claim 5 , wherein the fluorescent signal is generated by action of a polymerase having 5′-nuclease activity.
7. The method of claim 5 , wherein the oligonucleotide conjugate is a primer or a probe.
8. The method of claim 5 , further comprising the step of amplifying the target nucleic acid sequence.
9. A method for detecting a target nucleic acid sequence in a sample, comprising:
contacting the sample with an oligonucleotide conjugate having the formula:
wherein n is 1, 2, or 3,
wherein the oligonucleotide conjugate has a nucleic acid sequence at least partially complementary to the target nucleic acid sequence, and wherein the oligonucleotide conjugate further comprises a fluorophore; and
detecting a fluorescent signal resulting from hybridization of the oligonucleotide conjugate to the target nucleic acid sequence.
10. The method of claim 9 , wherein the fluorescent signal is generated by action of a polymerase having 5′-nuclease activity.
11. The method of claim 9 , wherein the oligonucleotide conjugate is a primer or a probe.
12. The method of claim 9 , further comprising the step of amplifying the target nucleic acid sequence.