Triplex probe compositions and methods for polynucleotide detection

• US 20060194222A1
• Filed: 10/20/2005
• Published: 08/31/2006
• Est. Priority Date: 10/20/2004
• Status: Abandoned Application

• Current Assignee: Stratagene California
• Original Assignee: Stratagene California
• Inventors: Sorge, Joseph A., Happe, Scott, Firmin, Andrew
• Application Number:
  US11/254,334
• Publication Number:
  US 20060194222A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 2537/119 Triple helix formation
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

The present invention provides composition and methods for the detection and measurement of target nucleic acids. The probes of the present invention, or triplex probes, comprise a complex of three oligonucleotide probes including: (1) a first oligonucleotide probe, (2) a second oligonucleotide probe, and (3) a bridging oligonucleotide probe. In most aspects of the invention, the first and second oligonucleotide probes preferentially hybridize to the bridging oligonucleotide in the absence of a target nucleic acid. The first oligonucleotide probe contains one member of an interactive pair of labels and the second oligonucleotide probe contains the other member of the interactive pair of labels. Separation of the first and second oligonucleotide probes (e.g., binding to target, cleavage of first, second, or bridging oligonucleotide) generates a detectable signal indicating the presence of a target nucleic acid.

Claims

1. An oligonucleotide probe complex, comprising:

a first oligonucleotide probe and a second oligonucleotide probe, wherein at least one of said first or second oligonucleotide probes binds to a target nucleic acid, wherein each of said first and second oligonucleotide probes comprise a member of an interactive pair of labels; and

a bridging oligonucleotide probe, wherein said bridging oligonucleotide probe binds to at least a portion of each of said first and second oligonucleotide probes, and wherein said members of the interactive pair of labels are in close proximity when bound to the bridging oligonucleotide probe.

2. The oligonucleotide probe complex of claim 1, wherein said interactive pair of labels comprises a fluorophore and a quencher.

3. The oligonucleotide probe complex of claim 2, wherein of one of said fluorophore or said quencher is attached to a 3′

nucleotide of said first oligonucleotide probe and the other of said fluorophore or said quencher is attached to a 5′

nucleotide of said second oligonucleotide probe.

4. The oligonucleotide probe complex of claim 1, wherein said first and/or second oligonucleotide probes have one or more nucleotides which do not hybridize to said bridging oligonucleotide.

5. The oligonucleotide probe complex of claim 1, wherein said interactive pair of labels are separated by between 0 and 5 nucleotides when bound to said bridging oligonucleotide.

6. The oligonucleotide probe complex of claim 2, wherein said fluorophore is selected from the group consisting of FAM, R110, TAMRA, R6G, CAL Fluor Red 610, CAL Fluor Gold 540, and CAL Fluor Orange 560.

7. The oligonucleotide probe complex of claim 2, wherein said quencher is selected from the group consisting of DABCYL, BHQ-1, BHQ-2, and BHQ-3.

8. The oligonucleotide probe complex of claim 1, wherein a detectable signal increases by at least 2 fold upon cleavage or hybridization of said first or second oligonucleotide probes to said target nucleic acid.

9. The oligonucleotide probe complex of claim 1, wherein a detectable signal increases by at least 3 fold upon cleavage or hybridization of said first or second oligonucleotide probes to said target nucleic acid.

10. A method of detecting a target nucleic acid in a sample, comprising the steps of:

contacting the sample with the oligonucleotide probe complex of claim 1;

and determining the presence of the target nucleic acid in said sample, wherein a change in intensity of a signal is indicative of the presence of the target nucleic acid.

11. A method of detecting a target nucleic acid in a sample, comprising the steps of:

(1) providing a PCR mixture comprising the probe of claim 1, a nucleic acid polymerase, a nuclease and a pair of primers;

(2) contacting the PCR mixture with the sample to produce a PCR sample mixture; and

(3) incubating the PCR sample mixture of step 2, to allow amplification of the target nucleic acid and cleavage of said first and/or second oligonucleotide probes with said nuclease, wherein generation of a detectable signal is indicative of the presence of the target nucleic acid in said sample.

12. The method of claim 11, wherein said nucleic acid polymerase substantially lacks 5′

to 3′

exonuclease activity.

13. The method of claim 11, wherein the nucleic acid polymerase is a DNA polymerase.

14. The method of claim 11, wherein the said nuclease is a FEN nuclease.

15. The method of claim 11, wherein said nuclease and said nucleic acid polymerase are contained in a single enzyme.

16. A composition comprising the oligonucleotide probe complex of claims 1.

17. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising the oligonucleotide probe complex of claim 1 and packaging material therefor.

18. An oligonucleotide probe complex, comprising:

a first oligonucleotide probe and a second oligonucleotide probe, wherein at least one of said first or second oligonucleotide probes binds to a target nucleic acid, wherein each of said first and second oligonucleotide probes comprise a member of an interactive pair of labels and wherein said first and/or second probe binds to said target nucleic acid through a primer region; and

a bridging oligonucleotide probe, wherein said bridging oligonucleotide probe binds to at least a portion of each of said first and second oligonucleotide probes, and wherein said member of the interactive pair of labels are in close proximity when bound to the bridging oligonucleotide probe.

19. A composition comprising the oligonucleotide probe complex of claims 18.

20. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising the oligonucleotide probe complex of claim 18 and packaging material therefor.

21. A method of detecting a target nucleic acid in a sample, comprising the steps of:

(1) providing a PCR mixture comprising the probe of claim 18, a nucleic acid polymerase, and a primer;

(2) contacting the PCR mixture with the sample to produce a PCR sample mixture; and

(3) incubating the PCR sample mixture of step 2, to allow amplification of the target nucleic acid, wherein generation of a detectable signal is indicative of a presence of the target nucleic acid in said sample.

22. An oligonucleotide probe complex, comprising:

a first oligonucleotide probe and a second oligonucleotide probe, wherein each of said first and second oligonucleotide probes comprise a member of an interactive pair of labels; and

a bridging oligonucleotide probe, wherein said bridging oligonucleotide probe binds to, at least a portion of, each of said first and second oligonucleotide probes, wherein said bridging oligonucleotide probe binds to a target nucleic acid through a primer region, and wherein said member of the interactive pair of labels are in close proximity when bound to the bridging oligonucleotide probe.

23. A composition comprising the oligonucleotide probe complex of claims 22.

24. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising the oligonucleotide probe complex of claim 22 and packaging material therefor.

25. A method of detecting a target nucleic acid in a sample, comprising the steps of:

(1) providing a PCR mixture comprising the probe of claim 22, a nucleic acid polymerase, a nuclease and a primer;

(2) contacting the PCR mixture with the sample to produce a PCR sample mixture; and

(3) incubating the PCR sample mixture of step 2 to allow amplification of the target nucleic acid and cleavage of said first and/or second oligonucleotide probes with said nuclease, wherein generation of a detectable signal is indicative of the presence of the target nucleic acid in said sample.

26. An oligonucleotide probe complex, comprising:

a first oligonucleotide probe and a second oligonucleotide probe, wherein each of said first and second oligonucleotide probes comprise a member of an interactive pair of labels; and

a bridging oligonucleotide probe, wherein said bridging oligonucleotide probe, binds to at least a portion of each of said first and second oligonucleotide probes, wherein said bridging oligonucleotide probe binds to a target nucleic acid, and wherein said member of the interactive pair of labels are in close proximity when bound to the bridging oligonucleotide probe.

27. A composition comprising the oligonucleotide probe complex of claims 26.

28. A kit for generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising the oligonucleotide probe complex of claim 26 and packaging material therefor.

29. A method of detecting a target nucleic acid in a sample, comprising the steps of:

(1) providing a PCR mixture comprising the probe of claim 26, a nucleic acid polymerase, a nuclease and a primer;

(2) contacting the PCR mixture with the sample to produce a PCR sample mixture; and

(3) incubating the PCR sample mixture of step 2, to allow amplification of the target nucleic acid and cleavage of said first and/or second oligonucleotide probes with said nuclease, wherein generation of a detectable signal is indicative of the presence of the target nucleic acid in said sample.