Quantitative amplification with a labeled probe and 3′ to 5′ exonuclease activity
This invention provides methods and kits for performing a quantitative amplification reaction. The method employs a polymerase enzyme and an enzyme having a 3′ to 5′ exonuclease activity that cleaves the 3′ oligonucleotide of the probe.
1. A method of quantifying a target nucleic acid in an amplification reaction, the method comprising
incubating a template comprising the target nucleic acid with: amplification primers, a probe having a 3′ nucleotide that is a mismatch to the target sequence, or two or more of the 3′ nucleotides that are mismatched, a polymerase, and an enzyme having 3′ to 5′ exonuclease activity under conditions in which
i) the amplification primers are extended by the polymerase to amplify the target nucleic acid;
ii) the probe specifically hybridizes to the target nucleic acid, wherein the 3′ nucleotide is cleaved from the probe; and
detecting the amount of cleavage product by determining the Ct, thereby quantifying the target nucleic acid.
2. The method of claim 1 , wherein is the enzyme having 3′ to 5′ exonuclease activity and the polymerase are the same polypeptide.
3. The method of claim 1 , wherein the 3′ to 5′ exonuclease activity is provided by a proofreading polymerase.
4. The method of claim 1 , wherein the 3′ to 5′ exonuclease activity is provided by a mutant error-correcting polymerase that lacks substantial polymerase activity.
5. The method of claim 1 , wherein the polymerase is a family B polymerase.
6. The method of claim 1 , wherein the probe comprises an abasic site at an internal position of the probe.
7. The method of claim 1 , wherein the probe is labeled with a detectable moiety at an internal nucleotide.
8. The method of claim 7 , wherein the internal nucleotide having the detectable label is within 10 nucleotides of the 3′ end of the probe.
9. The method of claim 7 , wherein the probe comprises an abasic site at an internal position of the probe.
10. The method of claim 1 , wherein the probe is labeled with a detectable moiety at the 3′ end.
11. The method of claim 1 , wherein the probe is double-labeled with two interacting moieties, wherein one of the interacting moieties is on the 3′ end.
12. The method of claim 11 , wherein the cleaved 3′ nucleotide is detected by detecting a change in fluorescence intensity.
13. The method of claim 11 , wherein the moiety on the 3′ nucleotide is a quencher and the second interacting moiety is a fluorescent label.
14. The method of claim 11 , wherein the moiety on the 3′ end is a fluorescent label and the second interacting moiety is a quencher.
15. The method of claim 11 , wherein the moiety on the 3′ end is a fluorescent label and the second interacting moiety is second fluorescent label.
16. The method of claim 1 , wherein the probe further comprises at least one phosphorothioate linkages.
17. The method of claim 16 , wherein the phosphorothioate linkage is between the 3′ nucleotide of the probe and the adjacent nucleotide.
18. The method of claim 1 , wherein the probe comprises a minor groove binding moiety.
19. The method of claim 1 , wherein the minor groove binding moiety is at the 5′ end of the probe.
20. A method of quantifying a target nucleic acid in an amplification reaction, the method comprising
incubating a template comprising the target nucleic acid with: amplification primers, a dual-labeled probe comprising a moiety at the 3′ end and a second interacting moiety, and a hot-start hybrid proofreading polymerase under conditions in which
i) the amplification primers are extended by the polymerase to amplify the target nucleic acid;
ii) the probe specifically hybridizes to the target nucleic acid, wherein the labeled 3′ end nucleotide is cleaved from the probe; and
detecting the amount of cleavage product by Ct, thereby quantifying the target nucleic acid.
21. The method of claim 20 , wherein the probe further comprises a minor groove binding moiety.
22. A method of quantifying a target nucleic acid in an amplification reaction, the method comprising
incubating a template comprising the target nucleic acid with: amplification primers, a dual-labeled probe comprising a moiety at the 3′ end and a second interacting moiety, a hot-start polymerase that lacks substantial 3′ to 5′ exonuclease activity, and a proofreading enzyme that lacks substantial polymerase activity under conditions in which
i) the amplification primers are extended by the polymerase that lacks substantial 3′ to 5′ exonuclease activity to amplify the target nucleic acid;
ii) the probe specifically hybridizes to the target nucleic acid, wherein the labeled 3′ end nucleotide is cleaved from the probe; and
detecting the amount of cleavage product by Ct, thereby quantifying the target nucleic acid.
23. The method of claim 22 , wherein the proofreading enzyme that lacks substantial polymerase activity is a variant family B polymerase that has an increased ratio of double-stranded exonuclease activity to single-stranded exonuclease activity where the increased ratio is relative to a parent family B polymerase from which the variant is derived.
24. The method of claim 22 , wherein the proofreading enzyme that lacks substantial polymerase activity is a family B polymerase that has a mutation in the YxGG domain or a mutation in the dNTP binding motif.
25. The method of claim 22 , wherein the probe further comprises a minor groove binding moiety.