Oligonucleotides labeled with a plurality of fluorophores

• US 7,667,024 B2
• Filed: 11/19/2004
• Issued: 02/23/2010
• Est. Priority Date: 11/19/2003
• Status: Expired due to Fees

• Current Assignee: Allelogic Biosciences Corporation
• Original Assignee: Allelogic Biosciences Corporation
• Inventors: Mao, Fei, Xin, Xing
• Primary Examiner(s): Strzelecka; Teresa E
• Application Number:
  US10/993,625
• Publication Number:
  US 20050272053A1
• Time in Patent Office: 1,922 Days
• Field of Search: None
• US Class Current: 536/24.3
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: Y10S 435/975 Kit

Abstract

An embodiment of the invention discloses new methods for designing labeled nucleic acid probes and primers by labeling oligonucleotides with a plurality of spectrally identical or similar dyes and optionally with one or more quencher dyes. Oligonucleotides labeled in accordance with some embodiments of the invention exhibit a detectable increase in signal, for example, fluorescent signal when the labeling dyes are separated from one another. Methods for separating the dye include cleaving the labeled oligonucleotides include using enzymes that have 5′-exonuclease activity. In one embodiment nucleic acid primers of the present invention may fluoresce upon hybridization to a target sequence and incorporation into the amplification product. Nucleic acid probes and primers of the present invention have wide applications ranging from general detection of a target nucleic acid sequence to clinical diagnostics. Major advantages of the oligonucleotides including nucleic acid probes and primers of many embodiments of the present invention are their synthetic simplicity, spectral versatility and superior fluorescent signal.

Claims

1. A probe comprising a nucleic acid sequence, said sequence having a 5′

end and a 3′

end, wherein the 3′

end is blocked to prevent extension via polymerization, and wherein the nucleic acid sequence is labeled with only two spectrally similar or identical fluorophores, wherein a first fluorophore is attached to a first site of said probe and a second fluorophore is attached to a second site of said probe, the first site and the second site being separated from one another by about 3 to about 60 nucleotides, and wherein said probe exhibits a random coil conformation when unhybridized with a target polynucleotide and yields an increase in detectable emission from the first fluorophore and the second fluorophore upon separation of the first or the second fluorophore from said probe.

2. The probe according to claim 1, wherein said probe further includes at least one cleavage site located between the two fluorophores attached to said probe.

3. The oligonucleotide probe according to claim 1, wherein the two fluorophores attached to said probe are separated from one another by 3 to 35 nucleotides.

4. The probe according to claim 1, wherein the two fluorophores attached to said probe are separated from one another by about 12 to about 35 nucleotides.

5. The probe according to claim 1, wherein absorption wavelengths of the two fluorophores are within 15 nm.

6. The probe according to claim 1, wherein the fluorophores are selected from the group consisting of a dye that has delocalized positive charge, a dye that has a delocalized negative charge, and a dye that has an equal number of positive and negative charges.

7. The probe according to claim 1, wherein said probe has a 5′

-end and a 3′

-end, wherein the first fluorophore is attached to the 5′

-end of said probe, and wherein the second fluorophore is attached to the 3′

end of said probe.

8. The probe according to claim 7, further including at least a first flexible aliphatic linker and a second aliphatic linker, wherein the first fluorophore is attached to the first linker and the first linker is attached to the 5′

-end of said probe, and the second fluorophore is attached to the second linker, and the second linker is attached to the 3′

end of said probe.

9. The probe according to claim 1, wherein said labeled probe is suitable in a polynucleotide amplification reaction performed at a temperature of about 56°

C. or higher.

10. The probe according to claim 9, wherein said polynucleotide amplification reaction is selected from the group consisting of PCR, multiplex PCR, real time PCR, quantitative PCR, and real time quantitative PCR.

19. A method of using a probe comprising the steps of:

supplying a sample of a target nucleic acid; and

hybridizing a probe of claim 1 to the target nucleic acid.

20. The method of using an probe according to claim 19, wherein said sample is selected from the group consisting of tissue extract, cell extract, bodily fluid, in vitro nucleic acid synthesis reaction, and PCR reaction mixture.

21. The method of using a probe according to claim 19, wherein said labeled probe is used in a nucleic acid amplification reaction wherein the reaction is selected form the group consisting of PCR, multiplex PCR, real-time PCR, quantitative PCR, and real-time quantitative PCR.

22. The method of using a probe according to claim 19, wherein said probe is for identifying the presence of a target sequence of nucleic acid polymer in the sample.

23. The method of using a probe according to claim 19, wherein said sample is applied to a nucleic acid chip and said nucleic acid chip includes said labeled probe.

24. The probe according to claim 6, wherein the dye having the delocalized positive charge is a cyanine dye.

25. The probe according to claim 6, wherein the dye having an equal number of positive and negative charges is a rhodamine dye.

26. The probe according to claim 7, wherein the fluorophores are identical.

27. The probe according to claim 9, wherein the fluorophores are identical.

28. The probe according to claim 1, wherein emission wavelengths of the two fluorophores are within 15 nm.

29. The probe according to claim 1, wherein the first or second fluorophore is a xanthene dye.

30. The probe according to claim 1, wherein the first or second fluorophore is a cyanine dye.

31. The probe according to claim 1, wherein the probe yields an increase in detectable emission from the fluorophores, which correlates with the amount of target polynucleotide amplified in an amplification reaction.

32. A kit comprising the probe of claim 1.

36. The kit of claim 32 comprising reagents for nucleic acid amplification and a polymerase possessing 5′

-exonuclease activity.

38. The probe of claim 1 wherein said probe is complementary to a region of the target polynucleotide.

39. The probe of claim 1 wherein at least one of the two spectrally similar fluorophores is a non-sulfonated dye.

40. The probe of claim 38 wherein the two spectrally similar fluorophores are non-sulfonated dyes.

41. The probe of claim 38 or 39 wherein the non-sulfonated dye is a xanthene dye.

42. The method of claim 38 or 39 wherein the non-sulfonated dye is a cyanine dye.

11. A method of labeling a probe having a 5′

end and a 3′

end, wherein the 3′

end is blocked to prevent extension via polymerization, wherein said probe is labeled with two spectrally similar or identical fluorophores, said method comprising;

attaching a first fluorophore to a first site of said probe sequence and a second fluorophore to a second site of said probe sequence, the first site and the second site being separated from one another by about 3 to about 60 nucleotides, thereby labeling said probe, such that said probe sequence exhibits a random coil conformation when unhybridized with a target polynucleotide and yields an increase in detectable emission from the first fluorophore and the second fluorophore upon separation of the first or the second fluorophore from said probe.

12. The method of labeling a probe according to claim 11, wherein the two fluorophores attached to said probe are separated from one another by about 3 to about 35 nucleotides.

13. The method of labeling a probe according to claim 11, wherein the two fluorophores attached to said probe are separated from one another by about 12 to about 35 nucleotides.

14. The method of labeling a probe according to claim 11, wherein absorption wavelengths of the two fluorophores are within 15 nm.

15. The method of labeling a probe according to claim 11, wherein at least one of the fluorophores is selected from the group consisting of a dye that has delocalized positive charge, a dye that has a delocalized negative charge, and a dye that has an equal number of positive and negative charges.

16. The method of labeling a probe according to claim 11, further including:

adding a plurality of flexible aliphatic linkers including a first linker and a second linker, wherein the first fluorophore is attached to the first linker, and the first linker is attached to the 5′

-end of said probe, and the second fluorophore is attached to the second linker, and the second linker is attached to the 3′

-end of said probe.

35. The method of claim 11, wherein the fluorophores are identical.

17. A kit for labeling a probe, the kit comprising:

a probe, said probe having a random coil conformation when unhybridized with a target polynucleotide, a length of at least 7 nucleotides and including at least a first site and a second site, said probe having a 5′

end and a 3′

end, wherein the 3′

end is blocked to prevent extension via polymerization;

two spectrally similar or identical fluorophores each having a reactive group, wherein a first fluorophore is reactive for attaching to the first site of said probe, and a second fluorophore is reactive for attaching to the second site of said probe such that the first fluorophore and the second fluorophores attached to said probe are separated from one another by about 3 to about 60 nucleotides, and wherein said probe when labeled yields an increase in detectable emission from the first fluorophore and the second fluorophore upon separation of the first or the second fluorophore from said probe.

18. The kit for labeling a probe according to claim 17, wherein at least one of the two fluorophores is selected from the group consisting of a dye that has a delocalized positive charge, a dye that has a delocalized negative charge, and a dye that has an equal number of positive and negative charges.

33. A kit according to claim 17, wherein the two spectrally similar or identical fluorophores are of different types of fluorophores.

34. A kit according to claim 33, wherein the two different types of fluorophores are selected from the group consisting of a dye that has a delocalized positive charge, a dye that has a delocalized negative charge, and a dye that has an equal number of positive and negative charges.

37. The kit of claim 17 further comprising an instruction manual for carrying out said labeling of said probe.