SINGLE NUCLEOTIDE POLYMORPHISM ANALYSIS OF HIGHLY POLYMORPHIC TARGET SEQUENCES

• US 20090087922A1
• Filed: 01/28/2008
• Published: 04/02/2009
• Est. Priority Date: 10/02/2003
• Status: Active Grant

• Current Assignee: Elitechgroup MDX LLC
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Dempcy, Robert O., Belousov, Yevgeniy, Vorobiev, Alexei, Lokhov, Sergey G.
• Granted Patent: US 7,718,374 B2
• Time in Patent Office: Days
• US Class Current: 436/501
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• 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

Abstract

Methods and probes are provided for the analysis of target sequences having two or more polymorphisms wherein one of the polymorphisms is to be distinguished and another polymorphism is to be masked.

Claims

1-14. -14. (canceled)

15. A method for distinguishing in a sample between wild-type, mutant and heterozygous target polynucleotides in a target polynucleotide sequence having at least two single nucleotide polymorphisms wherein a first polymorphism is to be distinguished and a second single nucleotide polymorphism is not distinguished, each of said first and second polymorphisms being in a probe region of said target polynucleotides, the method comprising:

(a) contacting said sample containing said target polynucleotide sequence with a first probe and a second probe that each distinguish a first polymorphism of interest, wherein the first probe preferentially hybridizes to the wild-type target polynucleotide and the second probe preferentially hybridizes to the mutant target polynucleotide, each of the first and second probes having an independently selected formula;

16. The method of claim 15, wherein the minor groove binder (M) is selected from the group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1-c][1,4]benzodiazepine analogs.

17. The method of claim 15, wherein the minor groove binder (M) is a substituted dihydrocyclopyrroloindole triamide (DPI₃).

18. The method of claim 15, wherein the minor groove binder (M) is attached to the 5′

end of the oligonucleotide conjugate

19. The method of claim 15, wherein the minor groove binder (M) is attached to the 3′

end of the oligonucleotide conjugate

20. The method of claim 15, wherein Q is a quencher with an absorption spectra between about 400 to 800 nm, and Fl is a fluorophore with emission wavelengths between about 400 to 800 nm

21. The method of claim 15, wherein said fluorophore (Fl) is selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs.

22. The method of claim 15, wherein said quencher (Q) is selected from the group consisting of mono azo and bis azo dyes

23. The method of claim 15, wherein said sugar phosphate backbone (A) comprises 8-25 nucleotides in length

24. The method of claim 15, wherein the fluorescence is produced directly upon hybridization of the probe with said target nucleic acid sequence.

25. The method of claim 15, wherein the fluorescence is produced upon enzymatic cleavage of the quencher or the fluorophore from the conjugate probe upon hybridization with a target nucleic acid sequence.

26. The method of claim 15, wherein the universal base is selected from the group provided in Table 1.

27. The method of claim 15, wherein the promiscuous base is selected from the group provided in Table 2

28. The method of claim 15, wherein the promiscuous (indiscriminative) base has the formula:

29. A method for distinguishing in a sample between wild-type, mutant and heterozygous target polynucleotides in a target polynucleotide sequence having at least two single nucleotide polymorphisms wherein a first polymorphism is to be distinguished and a second single nucleotide polymorphism is not distinguished, each of said first and second polymorphisms being in a probe region of said target polynucleotides, the method comprising:

(a) contacting said sample containing said target polynucleotide sequence with a first probe and a second probe that each distinguish a first polymorphism of interest, wherein the first probe preferentially hybridizes to the wild-type target polynucleotide and the second probe preferentially hybridizes to the mutant target polynucleotide, each of the first and second probes having an independently selected formula;

30. The method of claim 29, wherein the minor groove binder (M) is selected from the group consisting of CC1065 analogs, lexitropsins, distamycin, netropsin, berenil, duocarmycin, pentamidine, 4,6-diamino-2-phenylindole and pyrrolo[2,1-c][1,4]benzodiazepine analogs.

31. The method of claim 29, wherein the minor groove binder (M) is a substituted dihydrocyclopyrroloindole triamide (DPI₃).

32. The method of claim 29, wherein the minor groove binder (M) is attached to the 5′

end of the oligonucleotide conjugate

33. The method of claim 29, wherein the minor groove binder (M) is attached to the 3′

end of the oligonucleotide conjugate.

34. The method of claim 29, wherein Q is a quencher with an absorption spectra between about 400 to 800 nm, and Fl is a fluorophore with emission wavelengths between about 400 to 800 nm

35. The method of claim 29, wherein said fluorophore (Fl) is selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs.

36. The method of claim 29, wherein said quencher (Q) is selected from the group consisting of mono azo and bis azo dyes

37. The method of claim 29, wherein said sugar phosphate backbone (A) comprises 8-25 nucleotides in length

38. The method of claim 29, wherein the fluorescence is produced directly upon hybridization of the probe with said target nucleic acid sequence

39. The method of claim 29, wherein the fluorescence is produced upon enzymatic cleavage of the quencher or the fluorophore from the conjugate probe upon hybridization with a target nucleic acid sequence

40. The method of claim 29, wherein the universal base is selected from the group provided in Table 1

41. The method of claim 29, wherein the promiscuous base is selected from the group provided in Table 2

42. The method of claim 29, wherein the promiscuous (indiscriminative) base has the formula:

43. A method for distinguishing in a sample between wild-type, mutant and heterozygous target polynucleotides in a target polynucleotide sequence having at least two single nucleotide polymorphisms wherein a first polymorphism is to be distinguished and a second single nucleotide polymorphism is not distinguished, each of said first and second polymorphisms being in a probe region of said target polynucleotides, the method comprising:

(a) contacting a sample containing at least one target polynucleotide sequence that comprises one or more polymorphisms not intended to be distinguished with a probe that hybridizes to both said wild-type target and said mutant target polynucleotide, wherein said probe preferentially hybridizes with the wild-type target polynucleotide in comparison to the mutant target polynucleotide, the probe having a formula;

44-46. -46. (canceled)