Hybridization assay using self-quenching fluorescence probe

• US 6,258,569 B1
• Filed: 11/08/1999
• Issued: 07/10/2001
• Est. Priority Date: 11/16/1994
• Status: Expired due to Term

• Current Assignee: Perkinelmer Incorporated, Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perkinelmer Incorporated
• Inventors: Livak, Kenneth J., Mullah, Khairuzzaman Bashar, Flood, Susan J. A., Mamoro, Jeffrey
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/436,454
• Time in Patent Office: 610 Days
• Field of Search: 435/5, 435/6, 435/91.1, 435/91.2, 536/24.3, 536/24.33, 536/24.32, 536/26.6, 536/25.3, 536/25.32
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

Provided is a method of nucleic acid amplification. In one embodiment, the method comprises performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5′ to 3′ nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3′ relative to the primer and the probe does not hybridize with itself to form a hairpin structure. The oligonucleotide probe has at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe. Digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.

Claims

1. A method of nucleic acid amplification comprising:

performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5′

to 3′

nuclease activity, a primer capable of hybridizing to the target polynucleotide, and an oligonucleotide probe under amplification conditions such that the probe hybridizes to the target polynucleotide 3′

relative to the primer and the probe does not hybridize with itself to form a hairpin structure,

the oligonucleotide probe having at one end a fluorescent reporter and at the other end a quencher that quenches the fluorescence of the reporter molecule when both the fluorescent reporter and quencher are attached to the probe,

under conditions such that digestion of the oligonucleotide probe by the polymerase during amplification is effective to separate the reporter from the quencher, whereby a fluorescence signal of the reporter is increased.

2. The method of claim 1, wherein the polymerase is a thermostable nucleic acid polymerase.

3. The method of claim 1, wherein the reporter is a fluorescein dye and the quencher is a rhodamine dye.

4. The method of claim 3, wherein the fluorescent reporter is attached to the 5′

end of the probe.

5. The method of claim 4, wherein the probe is 15 to 60 nucleotides in length.

6. The method of claim 4, wherein the probe is 18 to 30 nucleotides in length.

7. The method of claim 3, wherein the fluorescent reporter is attached to the 3′

end of the probe.

8. The method of claim 7, wherein the probe is 15 to 60 nucleotides in length.

9. The method of claim 7, wherein the probe is 18 to 30 nucleotides in length.

10. The method of claim 1, wherein the fluorescent reporter is attached to the 5′

end of the probe.

11. The method of claim 10, wherein the probe is 15 to 60 nucleotides in length.

12. The method of claim 10, wherein the probe is 18 to 30 nucleotides in length.

13. The method of claim 1, wherein the fluorescent reporter is attached to the 3′

end of the probe.

14. The method of claim 13, wherein the probe is 15 to 60 nucleotides in length.

15. The method of claim 13, wherein the probe is 18 to 30 nucleotides in length.

16. The metod of claim 1, wherein the repoter or the quencher comprises a rhodamine dye.

17. The method of claim 1, wherein the reporter or the quencher comprises a fluorescein dye.

18. The method of claim 1, wberein the reporter or quencher is selected from the group consisting of a xanthene dye and a naphthylamine dye.

19. The method of claim 1, wherein the oligonucleotide probe is not capable of extension by the nucleic acid polymerase.

20. The method of claim 19, wherein the polymerase is a thermostable nucleic acid polymerase.

21. The method of claim 19, wherein the reporter is a fluorescein dye and the quencher is a rhodamine dye.

22. The method of claim 21, wherein the fluorescent reporter is attached to the 5′

end of the probe.

23. The method of claim 22, wherein the probe is 15 to 60 nucleotides in length.

24. The method of claim 22, wherein the probe is 18 to 30 nucleotides in length.

25. The method of claim 21, wherein the fluorescent reporter is attached to the 3′

end of the probe.

26. The method of claim 25, wherein the probe is 15 to 60 nucleotides in length.

27. The method of claim 25, wherein the probe is 18 to 30 nucleotides in length.

28. The method of claim 19, wherein the fluorescent reporter is attached to the 5′

end of the probe.

29. The method of claim 28, wherein the probe is 15 to 60 nucleotides in length.

30. The method of claim 28, wherein the probe is 18 to 30 nucleotides in length.

31. The method of claim 19, wherein the fluorescent reporter is attached to the 3′

end of tbe probe.

32. The method of claim 31, wherein the probe is 15 to 60 nucleotides in length.

33. The method of claim 31, wherein the probe is 18 to 30 nucleotides in length.

34. The method of claim 19, wherein the fluorescent reporter or the quencher comprises a rhodamine dye.

35. The method of claim 19, wherein the fluorescent reporter or the quencher comprises a fluorescein dye.

36. The method of claim 19, wherei dte reporter or quencher is selected from the group consisting of a xanthene dye and a naphthylamine dye.