Single nucleotide polymorphism analysis of highly polymorphic target sequences

• US 20050118623A1
• Filed: 09/29/2004
• Published: 06/02/2005
• 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 7,348,146 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 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. A method for continuous monitoring of polynucleotide amplification of a target nucleic acid sequence having at least two single nucleotide polymorphisms wherein a first single nucleotide polymorphism is to be distinguished and a second single nucleotide polymorphism is not distinguished, each of said polymorphisms being in a probe region of said target nucleic acid, the method comprising:

(a) combining a sample containing said target nucleic acid with one or more oligonucleotide primers adjacent to or overlapping with said probe region of the target sequence, a polymerizing enzyme, nucleotide substrates, and an oligonucleotide conjugate having a formula;

wherein M is a minor groove binder;

the subscript t is 0 or 1;

Q is a quencher;

W is a linking group;

K is a bond or a linking group;

Fl is a fluorophore; and

[A-B]_n represents a nucleic acid oligomer having n units, wherein n is an integer of from 5 to 50;

each A independently represents a nucleic acid backbone component selected from the group consisting of a sugar phosphate backbone, a modified sugar phosphate backbone, a locked nucleic acid backbone, a peptidic backbone or a variant thereof used in nucleic acid preparation; and

each B independently represents a nucleic acid base, a modified base or a base analog and wherein the base at the site complementary to said second single nucleotide polymorphism, is a universal or promiscuous (indiscriminative) base and the oligonucleotide portion has a sequence complementary to a portion of the target sequence being amplified, to provide a mixture;

(b) incubating the mixture under conditions favorable for polymerization; and

(c) continuously monitoring the amplification by monitoring the fluorescence produced upon conjugate hybridization to the amplified target.

2. The method of claim 1, 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.

3. The method of claim 1, wherein the minor groove binder (M) is a substituted dihydrocyclopyrroloindole triamide (DPI₃).

4. The method of claim 1, wherein the minor groove binder (M) is attacked to the 5′

end of the oligonucleotide conjugate.

5. The method of claim 1, wherein the minor groove binder (M) is attached to the 3′

end of the oligonucleotide conjugate.

6. The method of claim 1, 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.

7. The method of claim 1, wherein said fluorophore (Fl) is selected from the group consisting of coumarins, resorufins, xanthenes, benzoxanthenes, cyanines and bodipy analogs.

8. The method of claim 1, wherein said quencher (Q) is selected from the group consisting of mono azo and bis azo dyes.

9. The method of claim 1, wherein said sugar phosphate backbone (A) comprises 8-25 nucleotides in length.

10. The method of claim 1, wherein the fluorescence is produced directly upon hybridization of the probe with said target nucleic acid sequence.

11. The method of claim 1, 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.

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

13. The method of claim 1, wherein the promiscuous base is selected from the group provided in Table 2.

14. The method of claim 1, wherein the promiscuous (indiscriminative) base has the formula:

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;

wherein M is a minor groove binder;

the subscript t is 0 or 1;

Q is a quencher;

W is a linking group;

K is a bond or a linking group;

Fl is a fluorophore and each probe has a different fluorophore; and

[A-B]_n represents a nucleic acid oligomer having n units, wherein n is an integer of from 5 to 50;

each A independently represents a nucleic acid backbone component selected from the group consisting of a sugar phosphate backbone, a modified sugar phosphate backbone, a locked nucleic acid backbone, a peptidic backbone or a variant thereof used in nucleic acid preparation; and

each B independently represents a nucleic acid base, a modified base or a base analog and wherein the base at the site complementary to said second single nucleotide polymorphism, is a universal or promiscuous (indiscriminative) base, and the oligonucleotide portion has a sequence complementary to a portion of the target sequence being amplified, to provide a mixture;

(b) measuring the fluorescence produced on hybrid formation, whereby hybrid formation indicates the presence or absence of each of the wild-type, mutant and heterozygous target polynucleotides.

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;

wherein M is a minor groove binder;

the subscript t is 0 or 1;

Q is a quencher;

W is a linking group;

K is a bond or a linking group;

Fl is a fluorophore and each probe has a different fluorophore; and

[A-B]_n represents a nucleic acid oligomer having n units, wherein n is an integer of from 5 to 50;

each A independently represents a nucleic acid backbone component selected from the group consisting of a sugar phosphate backbone, a modified sugar phosphate backbone, a locked nucleic acid backbone, a peptidic backbone or a variant thereof used in nucleic acid preparation; and

each B independently represents a nucleic acid base, a modified base or a base analog and wherein the base at the site complementary to said second single nucleotide polymorphism, is a universal or promiscuous (indiscriminative) base and the oligonucleotide portion has a sequence complementary to a portion of the target sequence being amplified, to provide a mixture; and

(b) measuring the fluorescence using melting curve analysis of hybrid formation to determine the presence or absence of each of the wild-type, mutant and heterozygous target polynucleotides.

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 (O) 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;

wherein M is a minor groove binder;

the subscript t is 0 or 1;

Q is a quencher;

W is a linking group;

K is a bond or a linking group;

Fl is a fluorophore; and

[A-B]_n represents a nucleic acid oligomer having n units, wherein n is an integer of from 5 to 50;

each A independently represents a nucleic acid backbone component selected from the group consisting of a sugar phosphate backbone, a modified sugar phosphate backbone, a locked nucleic acid backbone, a peptidic backbone or a variant thereof used in nucleic acid preparation; and

each B independently represents a nucleic acid base, a modified base or a base analog and wherein the base at the site complementary to said second single nucleotide polymorphism, is a universal or promiscuous (indiscriminative) base and the oligonucleotide portion has a sequence complementary to a portion of the target sequence being amplified, to provide a mixture; and

(b) measuring the fluorescence produced on hybrid formation, whereby hybrid formation indicates the presence or absence of each of the wild-type, mutant and heterozygous target polynucleotides.

44. A compound selected from the group consisting of:

45. An oligonucleotide primer or probe prepared using a compound of claim 44.

46. An oligonucleotide primer containing one or more promiscuous bases selected from the group consisting of compounds 49, 50, 51, 52, 54 and 55 of Table 2.