Methods and compositions for nucleic acid amplification

• US 20030104421A1
• Filed: 05/07/2002
• Published: 06/05/2003
• Est. Priority Date: 05/07/2001
• Status: Abandoned Application

• Current Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Original Assignee: CuraGen Corporation (Celldex Therapeutics Incorporated)
• Inventors: Simons, Jan Fredik, Swirsding, Kendra, Colangelo, Christopher M.
• Application Number:
  US10/140,853
• Publication Number:
  US 20030104421A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

A plurality of oligonucleotides are provided having use in the amplification of target nucleic acid sequences. The oligonucleotides are used in simultaneous amplification of a multiplicity of nucleic acid sequences, providing a more balanced amplification of all target sequences. The oligonucleotides also provides a reaction system for the amplification of nucleic acid targets, and provide a method for selectively detecting one or more target nucleic acid sequences in a population of nucleic acid molecules, as well as a method for detecting a single nucleotide polymorphism in a population of nucleic acid molecules.

Claims

1. A plurality of oligonucleotides, the plurality comprising

(a) a first oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a B subsequence, wherein said B subsequence is provided at the 3′

terminus of said first oligonucleotide, and wherein the B subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence; and

(b) a second oligonucleotide having a 5′

terminus and a 3′

terminus, the second oligo nucleotide comprising said A subsequence at its 3′

terminus.

2. The plurality of claim 1, further comprising a first reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a B′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the B′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said B subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

3. The plurality of claim 1, wherein the first oligonucleotide includes said A subsequence at its 5′

terminus.

4. The plurality of claim 1, wherein the second oligonucleotide includes said A subsequence at its 5′

terminus.

5. The plurality of claim 1, wherein the second oligonucleotide comprises the nucleic acid sequence of SEQ ID NO:

9.

6. The plurality of claim 1, wherein the B subsequence comprises the nucleic acid sequence selected from the group consisting of SEQ ID NO:

1, 3, 5 and 7 and the B¹ subsequence comprises the nucleic acid sequence selected from the group consisting of SEQ ID NO;

2, 4, 6 and 8.

7. The plurality of claim 1, wherein the second oligonucleotide is present at a higher concentration in said plurality than said first oligonucleotide.

8. The plurality of claim 1, wherein the second oligonucleotide is present at 2, 5, 10, 15, 20, 50-fold higher molar concentration than said first oligonucleotide.

9. The plurality of claim 1, wherein the first oligonucleotide is 30-50 nucleotides in length.

10. The plurality of claim 1, wherein the first oligonucleotide is 35-45 nucleotides in length.

11. The plurality of claim 1, wherein the first oligonucleotide is about 40 nucleotides in length.

12. The plurality of claim 1, wherein the second oligonucleotide is 15-25 nucleotides in length.

13. The plurality of claim 1, wherein the second oligonucleotide is 17-23 nucleotides in length.

14. The plurality of claim 1, wherein the third oligonucleotide is 20 nucleotides in length.

15. The plurality of claim 1, further comprising a third oligonucleotide having a 5′

terminus and a 3′

terminus, the third oligonucleotide comprising said A subsequence at its 5′

terminus and a C subsequence at its 3′

terminus, wherein the C subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence.

16. The plurality of claim 1, further comprising a third reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a C′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the C′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said C subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

17. The plurality of claim 1, further comprising a fourth oligonucleotide having a 5′

terminus and a 3′

terminus, the fourth oligonucleotide comprising said A subsequence at its 5′

terminus and a D subsequence at its 3′

terminus, wherein the D subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence.

18. The plurality of claim 1, further comprising a fourth reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a D′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the D′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said D subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

19. The plurality of claim 1, further comprising a fifth oligonucleotide having a 5′

terminus and a 3′

terminus, the fifth oligonucleotide comprising said A subsequence at its 5′

terminus and an E subsequence at its 3′

terminus, wherein the E subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence.

20. The plurality of claim 1, further comprising a fifth reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and an E′

subsequence, wherein said B subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the E′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said E subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

21. A reaction system for selectively detecting one or more target nucleic acid sequences in a population of nucleic acid molecules, the reaction system comprising

(a) a population of starting nucleic acid molecules known to or suspected of containing at least one target nucleic acid sequence;

(b) a first oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a B subsequence, wherein said B subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the B subsequence hybridizes to said at least one target nucleic acid sequence and the A subsequence does not hybridize to said at least one target nucleic acid sequence; and

(c) a second oligonucleotide having a 5′

terminus and a 3′

terminus, the second nucleotide comprising said A subsequence at its 3′

terminus.

22. The reaction system of claim 21, further comprising a first reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a B′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the B′

subsequence hybridizes to said at least one target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said B subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

23. The reaction system of claim 21, wherein the first oligonucleotide includes said A subsequence at its 5′

terminus.

24. The reaction system of claim 21, wherein the second oligonucleotide includes said A subsequence at its 5′

terminus.

25. The reaction system of claim 21, wherein the second oligonucleotide is present at a higher concentration in said plurality than said first oligonucleotide.

26. The reaction system of claim 21, wherein the second oligonucleotide is present at 2, 5, 10, 15, 20, 50-fold higher molar concentration than said first oligonucleotide.

27. The reaction system of claim 21, wherein the first oligonucleotide is 30-50 nucleotides in length.

28. The reaction system of claim 21, wherein the first oligonucleotide is 35-45 nucleotides in length.

29. The reaction system of claim 21, wherein the first oligonucleotide is about 40 nucleotides in length.

30. The reaction system of claim 21, wherein the second oligonucleotide is 15-25 nucleotides in length.

31. The reaction system of claim 21, wherein the second oligonucleotide is 17-23 nucleotides in length.

32. The reaction system of claim 21, wherein the third oligonucleotide is 20 nucleotides in length.

33. The reaction system of claim 21, comprising a third oligonucleotide having a 5′

terminus and a 3′

terminus, the third oligonucleotide comprising said A subsequence at its 5′

terminus and a C subsequence at its 3′

terminus, wherein the C subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence.

34. The reaction system of claim 21, further comprising a third reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a C′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the C′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said C subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

35. The reaction system of claim 21, further comprising a fourth oligonucleotide having a 5′

terminus and a 3′

terminus, the fourth oligonucleotide comprising said A subsequence at its 5′

terminus and a D subsequence at its 3′

terminus, wherein the D subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence.

36. The reaction system of claim 21, further comprising a fourth reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a D′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the D′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said D subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

37. The reaction system of claim 21, further comprising a fifth oligonucleotide having a 5′

terminus and a 3′

terminus, the fifth oligonucleotide comprising said A subsequence at its 5′

terminus and an E subsequence at its 3′

terminus, wherein the E subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence.

38. The reaction system of claim 21, further comprising a fifth reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and an E′

subsequence, wherein said B subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the E′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said E subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

39. The reaction system of claim 21, further comprising a polymerase.

40. The reaction system of claim 39, wherein the polymerase is selected from the group consisting of {favored polymerase)

41. A kit for selectively detecting one or more target nucleic acid sequences in a population of nucleic acid molecules, the kit comprising

(a) a first oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a B subsequence, wherein said B subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the B subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence; and

(b) a second oligonucleotide having a 5′

terminus and a 3′

terminus, the second nucleotide comprising said A subsequence at its 3′

terminus.

42. The kit of claim 41, further comprising a first reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a B′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the B′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said B subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

43. The kit of claim 41, further comprising instructions for the kit.

44. The kit of claim 41, wherein the first oligonucleotide includes said A subsequence at its 5′

terminus.

45. The kit of claim 41, wherein the second oligonucleotide includes said A subsequence at its 5′

terminus

46. The kit of claim 41, wherein the second oligonucleotide is present at a higher concentration than said first oligonucleotide.

47. The kit of claim 41, wherein the second oligonucleotide is present at 2, 5, 10, 15, 20, 50-fold higher molar concentration than said first oligonucleotide.

48. The kit of claim 41, wherein the first oligonucleotide is 30-50 nucleotides in length.

49. The kit of claim 41, wherein the first oligonucleotide is 35-45 nucleotides in length.

50. The kit of claim 41, wherein the first oligonucleotide is about 40 nucleotides in length.

51. The kit of claim 41, wherein the second oligonucleotide is 15-25 nucleotides in length.

52. The kit of claim 41, wherein the second oligonucleotide is 17-23 nucleotides in length.

53. The kit of claim 41, wherein the third oligonucleotide is 20 nucleotides in length.

54. The kit of claim 41, further comprising a third oligonucleotide having a 5′

terminus and a 3′

terminus, the third oligonucleotide comprising said A subsequence at its 5′

terminus and a C subsequence at its 3′

terminus, wherein the C subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence.

55. The kit of claim 41, further comprising a third reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a C′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the C′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said C subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

56. The kit of claim 41, further comprising a fourth oligonucleotide having a 5′

terminus and a 3′

terminus, the fourth oligonucleotide comprising said A subsequence at its 5′

terminus and a D subsequence at its 3′

terminus, wherein the D subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence.

57. The kit of claim 56, further comprising a fourth reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a D′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the D′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said D subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

58. The kit of claim 56, further comprising a fifth oligonucleotide having a 5′

terminus and a 3′

terminus, the third oligonucleotide comprising said A subsequence at its 5′

terminus and an E subsequence at its 3′

terminus, wherein the E subsequence hybridizes to a target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence.

59. The kit of claim 58, further comprising a fourth reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and an E′

subsequence, wherein said B subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the E′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said E subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence.

60. A method for selectively detecting one or more target nucleic acid sequences in a population of nucleic acid molecules, the method comprising

(a) contacting a population of starting nucleic acid molecules known to or suspected of containing at least one target nucleic acid sequence with an effective amount of a first oligonucleotide and an effective amount of a first reverse oligonucleotide to form a primed first oligonucleotide complex and a primed first reverse oligonucleotide complex,

said first oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a B subsequence, wherein said B subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the B subsequence hybridizes to said target nucleic acid sequence and the A subsequence does not hybridize to said target nucleic acid sequence, thereby forming an annealed first oligonucleotide-target nucleotide complex;

said first reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a B′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the B′

subsequence hybridizes to a target nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said B subsequence, and the A subsequence does not hybridize to said target nucleic acid sequence, thereby forming an annealed first reverse oligonucleotide-target nucleotide complex;

(b) extending said annealed first oligonucleotide complex and annealed first reverse oligonucleotide complex with a polymerase to form a first extended oligonucleotide sequence and first reverse extended oligonucleotide sequence;

(c) combining said first extended oligonucleotide sequence and first reverse extended oligonucleotide sequence with an effective amount of a second oligonucleotide to form annealed second oligonucleotide complex, wherein said second oligonucleotide has a 5′

terminus and a 3′

terminus, the second nucleotide comprising said A subsequence at its 3′

terminus;

(d) extending said annealed second oligonucleotide-target nucleotide complex and annealed first reverse oligonucleotide-target nucleotide complex with a polymerase, thereby forming extended second oligonucleotide sequences; and

(e) detecting said extended second oligonucleotide sequences, thereby selectively amplifying one or more target nucleic acid sequences in a population of nucleic acid molecules.

61. The method of claim 60, further comprising contacting said starting population of nucleic acid molecules with said second oligonucleotide.

62. The method of claim 61, wherein said second oligonucleotide is present at a higher concentration than said first oligonucleotide.

63. A method for detecting a single nucleotide polymorphism in a population of nucleic acid molecules, the method comprising

(a) contacting a population of starting nucleic acid molecules known to or suspected of containing at least one polymorphic nucleic acid sequence with an effective amount of a first oligonucleotide and an effective amount of a first reverse oligonucleotide to form a primed first oligonucleotide complex and a primed first reverse oligonucleotide complex,

said first oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a B subsequence, wherein said B subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the B subsequence hybridizes to said polymorphic nucleic acid sequence and the A subsequence does not hybridize to said polymorphic nucleic acid sequence, thereby forming an annealed first oligonucleotide-polymorphic nucleotide complex;

said first reverse oligonucleotide having a 5′

terminus and a 3′

terminus and comprising an A subsequence and a B′

subsequence, wherein said B′

subsequence is provided at the 3′

terminus of said oligonucleotide, and wherein the B′

subsequence hybridizes to a polymorphic nucleic acid sequence and can be extended to form an extension product comprising a sequence complementary to said B subsequence, and the A subsequence does not hybridize to said polymorphic nucleic acid sequence, thereby forming an annealed first reverse oligonucleotide-target nucleotide complex;

(b) extending said annealed first oligonucleotide complex and annealed first reverse oligonucleotide complex with a polymerase to form a first extended oligonucleotide sequence and first reverse extended oligonucleotide sequence;

(c) combining said first extended oligonucleotide sequence and first reverse extended oligonucleotide sequence with an effective amount of a second oligonucleotide to form annealed second oligonucleotide complex, wherein said second oligonucleotide has a 5′

terminus and a 3′

terminus, the second nucleotide comprising said A subsequence at its 3′

terminus;

(d) extending said annealed second oligonucleotide-polymorphic nucleotide complex and annealed first reverse oligonucleotide-target nucleotide complex with a polymerase, thereby forming extended second oligonucleotide sequences; and

(e) detecting said extended second oligonucleotide sequences, thereby identifying a single nucleotide polymorphic sequence in a population of nucleic acid molecules.

64. The method of claim 63, further comprising contacting said starting population of nucleic acid molecules with said second oligonucleotide.

65. The method of claim 63, wherein said second oligonucleotide is present at a higher concentration than said first oligonucleotide.