Methods of using FET labeled oligonucleotides that include a 3'-5' exonuclease resistant quencher domain and compositions for practicing the same

• US 20030165920A1
• Filed: 08/15/2002
• Published: 09/04/2003
• Est. Priority Date: 02/27/2002
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Spasic, Dragan, Chou, Quin
• Granted Patent: US 7,160,997 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

Methods and compositions are provided for detecting a primer extension product in a reaction mixture. In the subject methods, a primer extension reaction is conducted in the presence of a polymerase having 3′→5′ exonuclease activity and at least one FET labeled oligonucleotide probe that includes a 3′→5′ exonuclease resistant quencher domain. Also provided are systems and kits for practicing the subject methods. The subject invention finds use in a variety of different applications, and are particularly suited for use in high fidelity PCR based reactions, including SNP detection applications, allelic variation detection applications, and the like.

Claims

1. A method for detecting the production of a primer extension product in a primer extension reaction mixture, said method comprising:

(a) producing a primer extension mixture that includes a nucleic acid polymerase, a FET labeled oligonucleotide that includes a 3′

→

5′

exonuclease resistant quencher domain, and at least one of;

(i) a nucleic acid intercalator, and

(ii) a minor groove binder;

(b) subjecting said primer extension mixture to primer extension reaction conditions;

(c) detecting a change in a fluorescent signal from said FET labeled oligonucleotide probe to obtain an assay result; and

(d) employing said assay result to determine whether a primer extension product is present in said mixture.

2. The method according to claim 1, wherein said primer extension reaction is a PCR amplification reaction.

3. The method according to claim 2, wherein said method is a real-time method of monitoring said PCR amplification reaction.

4. The method according to claim 1, wherein said FET labeled oligonucleotide is a nucleic acid detector molecule that includes a single-stranded target binding sequence linked to fluorophore and dark quencher.

5. The method according to claim 4, wherein energy transfer occurs between said fluorophore and dark quencher of said FET labeled oligonucleotide probe upon fluorophore excitation when said FET labeled oligonucleotide is not hybridized to target nucleic acid.

6. The method according to claim 5, wherein energy transfer does not occur between said fluorophore and dark quencher of said FET labeled oligonucleotide probe upon fluorophore excitation when said FET labeled oligonucleotide probe is hybridized to a target nucleic acid.

7. The method according to claim 3, wherein said method is a 5′

nuclease method of monitoring a PCR amplification reaction.

8. The method according to claim 7, wherein energy transfer does not occur between said fluorophore and dark quencher of said FET labeled oligonucleotide probe upon fluorophore excitation when said FET labeled oligonucleotide probe is cleaved by 5′

nuclease.

9. The method according to claim 4, wherein said target binding sequence comprises a hybridization domain complementary to a sequence of said, primer extension product.

10. The method according to claim 1, wherein said primer extension mixture includes a nucleic acid intercalator.

11. The method according to claim 1, wherein said nucleic acid intercalator is bonded to said FET labeled oligonucleotide.

12. The method according to claim 11, wherein said nucleic acid intercalator is covalently bonded to said FET labeled oligonucleotide.

13. The method according to claim 10, wherein said nucleic acid intercalator provides increased stability to the hybrid formed from said FET labeled oligonucleotide.

14. The method according to claim 10, wherein said nucleic acid intercalator provides exonuclease activity resistance to said FET labeled oligonucleotide.

15. The method according to claim 10, wherein said nucleic acid intercalator comprises a polycyclic compound.

16. The method according to claim 15, wherein said polycyclic compound comprises an aromatic ring.

17. The method according to claim 15, wherein said polycyclic compound comprises at least three rings and not more than six rings.

18. The method according to claim 15, wherein said polycyclic compound comprises at least three rings, wherein at least two of said rings are fused.

19. The method according to claim 18, wherein said polycyclic compound is an acridine.

20. The method according to claim 1, wherein said primer extension mixture includes a minor groove binder.

21. The method according to claim 20, wherein said minor groove binder is present in the aqueous buffer medium of said primer extension mixture.

22. The method according to claim 20, wherein said minor groove binder is bound to said FET labeled oligonucleotide.

23. The method according to claim 20, wherein said minor groove binder provides increased stability to the hybrid formed from said FET labeled oligonucleotide.

24. The method according to claim 20, wherein said minor groove binder comprises a compound capable of binding within the minor groove of double stranded DNA.

25. The method according to claim 20, wherein said minor groove binder comprises a compound capable of binding within the minor groove of double stranded DNA with an association constant of at least 10³M⁻

1

26. The method according to claim 20, wherein said minor groove binder is a compound selected from the group consisting of netropsin, distamycin, lexitropsin, mithramycin, chromomycin A₃, olivomycin, anthramycin, sibiromycin, pentamidine, stilbamidine, berenil, CC-1065, Hoechst 33258, 4′

-6-diamidino-2-phenylindole (DAPI), and derivatives thereof.

27. The method according to claim 1, wherein said primer extension mixture includes a nucleic acid intercalator and a minor groove binder.

28. The method according to claim 1, wherein mixture includes a nucleic acid polymerase having 3′

→

5′

exonuclease activity.

29. A FET labeled probe comprising nucleic acid intercalator bonded to a FET labeled oligonucleotide.

30. The FET labeled probe according to claim 29, wherein said intercalator covalently bonded to said FET labeled oligonucleotide.

31. The FET labeled probe according to claim 29, wherein said nucleic acid intercalator is located at the 3′

end of said FET labeled oligonucleotide.

32. The FET labeled probe according to claim 29, wherein said nucleic acid intercalator is located at the 5′

end of said FET labeled oligonucleotide.

33. The FET labeled probe according to claim 29, wherein said nucleic acid intercalator provides increased stability to the hybrid formed from said FET labeled oligonucleotide.

34. The FET labeled probe according to claim 29, wherein said nucleic acid intercalator provides exonuclease activity resistance to said FET labeled oligonucleotide.

35. The FET labeled probe according to claim 29, wherein said nucleic acid intercalator comprises a polycyclic compound.

36. The FET labeled probe according to claim 35, wherein said polycyclic compound comprises an aromatic ring.

37. The FET labeled probe according to claim 35, wherein said polycyclic compound comprises at least three rings and not more than six rings.

38. The FET labeled probe according to claim 35, wherein said polycyclic compound comprises at least three rings, wherein at least two of said rings are fused.

39. The FET labeled probe according to claim 38, wherein said polycyclic compound is an acridine.

40. The FET labeled probe according to claim 29, wherein said FET labeled oligonucleotide is a nucleic acid detector molecule that includes a single-stranded target binding sequence linked to fluorophore and dark quencher.

41. The FET labeled probe according to claim 40, wherein said FET labeled oligonucleotide is a probe selected from the group consisting of:

Taqman probes, scorpion probes, sunrise probes, molecular beacons, conformationally assisted probes, and in situ hybridization probes.

42. A method of monitoring of a PCR amplification reaction, said method comprising:

(a) preparing a PCR amplification reaction mixture by combining;

(i) a template nucleic acid;

(ii) forward and reverse nucleic acid primers;

(iii) deoxyribonucleotides;

(iv) a nucleic acid polymerase;

(v) a FET labeled oligonucleotide that includes;

a 3′

→

5′

exonuclease resistant quencher domain comprising a dark quencher, a fluorescent reporter, domain comprising a fluorophore and a PCR product complementary domain; and

(vi) (vi) at least one of;

(A) a nucleic acid intercalator, and

(B) a minor groove binder;

(b) subjecting said PCR amplification reaction mixture to PCR amplification conditions;

(c) monitoring said reaction mixture for a fluorescent signal from said FET labeled oligonucleotide probe to obtain an assay result; and

(d) employing said assay result to monitor said PCR amplification reaction.

44. The method according to claim 42, wherein said method is a method of monitoring a PCR amplification reaction in real time.

45. The method according to claim 42, wherein said FET labeled oligonucleotide is a probe selected from the group consisting of:

scorpion probes, sunrise probes, molecular beacons, and conformationally assisted probes.

46. A method for screening a nucleic acid sample for the presence of first and second nucleic acids that differ from each other by a single nucleotide, said method comprising:

(a) producing a primer extension mixture that includes;

(i) said nucleic acid sample;

(ii) a nucleic acid polymerase;

(iii) first and second FET labeled oligonucleotide probes that are complementary to said first and second nucleic acids, respectively, wherein each of said first and second FET labeled oligonucleotides includes a 3′

→

5′

exonuclease resistant quencher domain; and

;

(iv) at least one of;

(A) a nucleic acid intercalator, and

(B) a minor groove binder

(b) subjecting said primer extension mixture to primer extension reaction conditions;

(c) detecting a change in a fluorescent signal, if any, from said first and second FET labeled oligonucleotide probes to obtain an assay result; and

(d) employing said assay result to determine the presence or absence of said first and second nucleic acids in said sample.

47. The method according to claim 46, wherein mixture includes a nucleic acid polymerase having 3′

→

5′

exonuclease activity.

48. A system for use in detecting the production of a primer extension product in a primer extension reaction mixture, said system comprising:

(a) a FET labeled oligonucleotide that includes a 3′

→

5′

exonuclease resistant quencher domain;

(b) a nucleic acid polymerase; and

(c) at least one of;

(i) a nucleic acid intercalator; and

(ii) a minor groove binder.

49. A kit for use in detecting the production of a primer extension product in a primer extension reaction mixture, said kit comprising:

(a) a FET labeled oligonucleotide that includes a 3′

→

5′

exonuclease resistant quencher domain;

(b) at least one of;

(i) a nucleic acid intercalator; and

(ii) a minor groove binder.

50. The kit according to claim 49, wherein said nucleic acid intercalator is covalently bonded to said FET labeled oligonucleotide.

51. The kit according to claim 49, wherein said kit includes both of said nucleic acid intercalator and said minor groove binder.

52. The kit according to claim 51, wherein said intercalator, said FET labeled oligonucleotide and said minor groove binder are present as a single composition.

53. The kit according to claim 52, wherein said intercalator is covalently bonded to said FET labeled oligonucleotide.

54. In a method comprising employing fluorescently labeled nucleic acid detector and a polymerase, the improvement comprising:

employing a FET labeled oligonucleotide that includes a 3′

→

5′

exonuclease resistant quencher domain as said fluorescently labeled nucleic acid detector, and at least one of;

(a) a nucleic acid intercalator; and

(b) a minor groove binder.