Format of Probes to Detect Nucleic Acid Differences

• US 20110039264A1
• Filed: 08/09/2010
• Published: 02/17/2011
• Est. Priority Date: 08/14/2009
• Status: Active Grant

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Will, Stephen G.
• Granted Patent: US 8,409,802 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2535/131 Allele specific probes

Abstract

The invention provides, inter alia, novel probes, methods, reaction mixtures, and kits for detecting the presence or absence of a target nucleic acid sequence.

Claims

1. A method of detecting the presence or absence of a target nucleic acid sequence in a biological sample, the method comprising:

a. contacting a detectably-labeled probe comprising an anchor nucleic acid domain and a reporter nucleic acid domain with the sample; and

b. detecting the presence or absence of binding of the probe to the target nucleic acid,

wherein the anchor and reporter domains are linked by a non-nucleoside linker, and neither the anchor nor the reporter domain forms a stem loop in the absence of the target nucleic acid;

and wherein

(i) the probe is not extendible by a polymerase; and

/or

(ii) the linker is linked to the anchor domain within 2 nucleotides of the 3′

end of the anchor domain and the linker is linked to the reporter domain within 2 nucleotides of the 5′

end of the reporter domain, wherein the anchor domain is not linked to a detectable label; and

/or

(iii) the anchor domain and the reporter domain each comprise a contiguous sequence of at least 6 nucleotides complementary to the same strand of the target nucleic acid.

2. The method of claim 1, wherein the probe is not extendible by a polymerase.

3. The method of claim 1, wherein the linker is linked to the anchor domain within 2 nucleotides of the 3′

end of the anchor domain and the linker is linked to the reporter domain within 2 nucleotides of the 5′

end of the reporter domain, wherein the anchor domain is not linked to a detectable label.

4. The method of claim 1, wherein the anchor domain and the reporter domain each comprise a contiguous sequence of at least 10 nucleotides complementary to one strand of the target nucleic acid. .

5. The method of claim 1, wherein the detecting step comprises measuring the melting temperature of a complex formed between the reporter domain and the target nucleic acid.

6. The method of claim 1, wherein the length of the reporter domain is between 4 to 20 nucleotides.

7. The method of claim 1, wherein the anchor region is between 6-40 nucleotides.

8. The method of claim 9, further comprising contacting the probe and sample with a soluble intercalating quencher such that the quencher alters fluorescence from the label when the reporter domain forms a complex with the target nucleic acid.

9. The method of claim 12, wherein the human nucleic acid comprises a single nucleotide polymorphism (SNP) and the reporter domain is 100% complementary to one allele of the SNP.

10. The method of claim 1, wherein the probe comprises at least one non-natural nucleotide, wherein the non-natural nucleotide increases the melting temperature of the reporter domain compared to a corresponding natural nucleotide in the place of the non-natural nucleotide.

11. The method of claim 1, wherein the linker is polyethylene glycol.

12. The method of claim 11, wherein the linker is hexa-ethylene glycol.

13. A reaction mixture for detecting the presence or absence of a target sequence comprising:

a. a target nucleic acid comprising an anchor binding region and a reporter binding region, and

b. a detectably-labeled probe comprising an anchor nucleic acid domain and a reporter nucleic acid domain,

wherein the anchor and reporter domains are linked by a non-nucleoside linker, and neither the anchor nor the reporter domain forms a stem loop in the absence of the target nucleic acid, and

wherein

(i) the probe is not extendible by a polymerase;

(ii) the linker is linked to the anchor domain within 2 nucleotides of the 3′

end of the anchor domain and the linker is linked to the reporter domain within 2 nucleotides of the 5′

end of the reporter domain, wherein the anchor domain is not linked to a detectable label; and

/or

(iii) the anchor domain and the reporter domain each comprise a contiguous sequence of at least 6 nucleotides complementary to the same strand of the target nucleic acid.

14. The reaction mixture of claim 13, further comprising nucleoside triphosphates, a DNA polymerase, and/or an oligonucleotide primer.

15. The reaction mixture of claim 13, wherein the probe is not extendible by a polymerase.

16. The reaction mixture of claim 13, wherein the length of the reporter domain is between 4 to 20 nucleotides.

17. The reaction mixture of claim 13, wherein the anchor region is between 6-40 nucleotides.

18. The reaction mixture of claim 13, further comprising a soluble intercalating quencher, wherein the quencher alters fluorescence from the label when the reporter domain forms a complex with the target nucleic acid.

19. The reaction mixture of claim 13, wherein the human nucleic acid comprises a single nucleotide polymorphism (SNP) and the reporter domain is 100% complementary to one allele of the SNP.

20. The reaction mixture of claim 13, wherein the probe comprises at least one non-natural nucleotide, wherein the non-natural nucleotide increases the melting temperature of the reporter domain compared to a corresponding natural nucleotide in the place of the non-natural nucleotide.

21. The reaction mixture of claim 13, wherein the linker is polyethylene glycol.

22. The reaction mixture of claim 21, wherein the linker is hexa-ethylene glycol.

23. A detectably-labeled probe comprising an anchor nucleic acid domain and a reporter nucleic acid domain, wherein:

the anchor and reporter domains are linked by a non-nucleoside linker;

neither the anchor nor the reporter domain forms a stem loop in the absence of the target nucleic acid; and

wherein;

(i) the probe is not extendible by a polymerase; and

/or

(ii) the linker is linked to the anchor domain within 2 nucleotides of the 3′

end of the anchor domain and the linker is linked to the reporter domain within 2 nucleotides of the 5′

end of the reporter domain, wherein the anchor domain is not linked to a detectable label.

24. The probe of claim 23, wherein the probe is not extendible by a polymerase.

25. The probe of claim 23, wherein the linker is linked to the anchor domain within 2 nucleotides of the 3′

end of the anchor domain and the linker is linked to the reporter domain within 2 nucleotides of the 5′

end of the reporter domain, wherein the anchor domain is not linked to a detectable label

26. The probe of claim 23, wherein the length of the reporter domain is between 4 to 20 nucleotides.

27. The probe of claim 23, wherein the anchor region is between 6-40 nucleotides.

28. The probe of claim 23, wherein the probe comprises at least one non-natural nucleotide, wherein the non-natural nucleotide increases the melting temperature of the reporter domain compared to a corresponding natural nucleotide in the place of the non-natural nucleotide.

29. The probe of claim 23, wherein the linker is polyethylene glycol

30. The probe of claim 29, wherein the linker is hexa-ethylene glycol.

31. A kit for detecting the presence or absence of a target sequence, the kit comprising:

a. a probe according to claim 23; and

b. one or more reagents selected from the group consisting of a salt, a buffer, a nuclease inhibitors, nucleoside triphosphates, a DNA polymerase, and/or an oligonucleotide primer.

32. The kit of claim 31, wherein the kit further comprises a soluble intercalating quencher such that the quencher alters fluorescence from the label when the reporter domain forms a complex with the target nucleic acid.

33. The kit of claim 31, wherein the probe is not extendible by a polymerase.

34. The kit of claim 31, wherein the length of the reporter domain is between 4 to 20 nucleotides.

35. The kit of claim 31, wherein the anchor region is between 6-40 nucleotides.

36. The kit of claim 31, wherein the probe comprises at least one non-natural nucleotide, wherein the non-natural nucleotide increases the melting temperature of the reporter domain compared to a corresponding natural nucleotide in the place of the non-natural nucleotide.