Detecting and amplifying target nucleic acids using exonucleolytic activity

• US 5,573,907 A
• Filed: 08/04/1993
• Issued: 11/12/1996
• Est. Priority Date: 01/26/1990
• Status: Expired due to Term

• Current Assignee: Abbott Laboratories Incorporated
• Original Assignee: Abbott Laboratories Incorporated
• Inventors: Rinehardt, Laurie A., Pabich, Edward K., Carrino, John J., Spies, Uwe
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): MYERS, CARLA J
• Application Number:
  US08/101,877
• Time in Patent Office: 1,196 Days
• Field of Search: 435/6, 435/91.2, 435/91.31, 435/91.52, 435/6, 435/91.1, 935/77, 935/78, 536/22.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/6.18
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• 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 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

The present invention relates to improved LCR amplification schemes using at least one downstream probe modified at its 5'"'"' end to reduce or eliminate target independent amplification. The different modified probes, and kits containing them are also presented. Also presented is a method for detecting differences in nucleic acid sequences, with reduced target independent amplification, using the modified probes.

Claims

1. A method for assaying a target nucleic acid sequence comprising the steps of:

(a) under hybridizing conditions exposing a sample suspected of containing the target nucleic acid sequence in single stranded form to an excess of a first set of oligonucleotides comprising a first upstream probe and a first downstream probe, both probes having sequences substantially complementary to portions of a target nucleic acid sequence, the 3'"'"' terminus of the first upstream probe hybridizing proximate to the 5'"'"' terminus of the first downstream probe, wherein the 5'"'"' end of the first downstream probe is modified to be ligation incompetent absent correction, thereby hybridizing the first set of oligonucleotides to the target nucleic acid sequence, if present;

(b) correcting the 5'"'"' end of the downstream probe when the downstream probe is hybridized to target, said correction including nucleolytic degradation of said 5'"'"' end, whereby the correction renders this 5'"'"' end ligation competent;

(c) ligating the corrected downstream probe to the upstream probe to form a ligated product; and

(d) determining to what extent the correction and ligation steps occur as a measure of the target nucleic acid in the sample.

2. The method of claim 1, wherein determining the extent of correction and ligation comprises separating ligated product from unligated probes and determining the amount of ligated product formed.

3. The method of claim 1, wherein determining the extent of correction and ligation comprises monitoring the release of cleaved fragments from the 5'"'"' end of said downstream probe, wherein said cleaving is performed by a template-dependent polymerase having 5'"'"' to 3'"'"' nucleolytic activity.

4. The method of claim 1, wherein said ligation incompetent 5'"'"' end comprises a non-phosphorylated 5'"'"' terminus and wherein said correction step comprises cleaving the terminal nucleoside to create a new 5'"'"' phosphorylated terminus on said downstream probe.

5. The method of claim 4, wherein said correction step further comprises extending the 3'"'"' terminus of said upstream probe by the addition of one or more nucleotide triphosphates in a template-dependent manner to bring said extended 3'"'"' terminus adjacent to said newly created 5'"'"' phosphorylated terminus.

6. The method of claim 5, wherein both said cleaving and extending steps are performed by a template-dependent polymerase having 5'"'"' to 3'"'"' nucleolytic activity.

7. The method of claim 4, wherein determining the extent of correction and ligation comprises separating ligated product from unligated probes and determining the amount of ligated product formed.

8. The method of claim 4, wherein said terminal nucleotide of the downstream probe carries a label, and wherein said cleaving is performed by a template-dependent polymerase having 5'"'"' to 3'"'"' nucleolytic activity and further wherein said determining the extent of correction and ligation comprises monitoring the release of label from the downstream probe.

9. The method of claim 8 wherein said label is a fluorescent label and said monitoring comprises fluorescence polarization.

10. The method of claim 1, wherein the ligation incompetent 5'"'"' end comprises at least one nucleotide base in said 5'"'"' end which is mismatched with respect to the target sequence to which it hybridizes, and wherein said correction step comprises cleaving the mismatched nucleotide to create a new 5'"'"' phosphorylated terminus on said downsteam probe.

11. The method of claim 10, wherein said correction step further comprises extending the 3'"'"' terminus of said upstream probe by the addition of one or more nucleotide triphosphates in a template-dependent manner to bring said extended 3'"'"' terminus adjacent to said newly created 5'"'"' phosphorylated terminus.

12. The method of claim 11, wherein both said cleaving and extending steps are performed by a template-dependent polymerase having 5'"'"' to 3'"'"' nucleolytic activity.

13. The method of claim 10, wherein said at least one mismatched base is positioned at the 5'"'"' terminal nucleotide.

14. The method of claim 10, wherein said at least one mismatched base is positioned within 1 to about 5 nucleotides internal of said 5'"'"' terminal nucleotide.

15. The method of claim 13 or 14, wherein said cleaving step includes cleaving a nucleotide adjacent the mismatched nucleotide on its 3'"'"' side.

16. The method of claim 10, wherein the ligation incompetent 5'"'"' end of said downstream probe further comprises a non-phosphorylated 5'"'"' terminus.

17. The method of claim 10, wherein determining the extent of correction and ligation comprises separating ligated product from unligated probes and determining the amount of ligated product formed.

18. The method of claim 10, wherein said cleaving is performed by a template-dependent polymerase having 5'"'"' to 3'"'"' nucleolytic activity, and wherein the cleaved portion of the 5'"'"' end of the downstream probe carries a label, and further wherein said determining the extent of correction and ligation comprises monitoring the release of label from the downstream probe.

19. The method of claim 18 wherein said label is a fluorescent label and said monitoring comprises fluorescence polarization.

20. The method of claim 10, wherein the upstream and downstream probes hybridize to target such that the mismatched 5'"'"' end of downstream probe overlaps the 3'"'"' end of the upstream probe by at least one overlapping base, and wherein the correction step comprises removal of said at least one overlapping base to create a new 5'"'"' phosphorylated terminus such that the newly created 5'"'"' terminus of the downstream probe abuts the 3'"'"' end of the upstream probe without extending the upstream probe, so that the two probes can be directly ligated.

21. The method of claim 20, wherein the mismatched 5'"'"' end of said downstream probe further comprises a non-phosphorylated 5'"'"' terminus.

22. The method of claim 1, further comprising an excess of a second set of oligonucleotides comprising a second upstream probe and a second downstream probe, both probes having sequences substantially complementary to the first downstream probe and first upstream probes, respectively, the 3'"'"' terminus of the second upstream probe being hybridized proximate to the 5'"'"' terminus of the second downstream probe, and

wherein said hybridization, correction and ligation steps (a-c) are repeated to effect an amplification of the target nucleic acid sequence.

23. The method of claim 22, wherein the ligation incompetent 5'"'"' end of said first downstream probe comprises a non-phosphorylated 5'"'"' terminus and wherein said correction step comprises cleaving the terminal nucleoside to create a new 5'"'"' phosphorylated terminus on said downstream probe.

24. The method of claim 23, wherein the 5'"'"' end of the second downstream probe is also modified to be ligation incompetent absent correction, and wherein said correction step includes nucleolytic degradation of the 5'"'"' end of said second downstream probe to create a new phosphorylated 5'"'"' terminus, whereby the correction step renders the 5'"'"' ends of both downstream probes ligation competent.

25. The method of claim 24, wherein said modification of the second downstream probe is selected from:

(a) a non-phosphorylated 5'"'"' terminus; and

(b) at least one nucleotide base in the 5'"'"' end which is mismatched with respect to the template sequence to which it hybridizes; and

wherein said correcting step includes cleaving said non-phosphorylated nucleotide or said mismatched nucleotide.

26. The method of claim 24, wherein said correction step further comprises extending the 3'"'"' terminus of both said upstream probes by the addition of one or more nucleotide triphosphates in a template-dependent manner to bring said extended 3'"'"' termini adjacent to said newly created 5'"'"' phosphorylated termini.

27. The method of claim 26, wherein both said cleaving and extending steps are performed by a template-dependent polymerase having 5'"'"' to 3'"'"' nucleolytic activity.

28. The method of claim 24, wherein determining the extent of correction and ligation comprises separating ligated product from unligated probes and determining the amount of ligated product formed.

29. The method of claim 24, wherein said terminal nucleotide of the downstream probe carries a label, and wherein said cleaving is performed by a template-dependent polymerase having 5'"'"' to 3'"'"' nucleolytic activity and further wherein said determining the extent of correction and ligation comprises monitoring the release of label from the downstream probe.

30. The method of claim 22, wherein the ligation incompetent 5'"'"' end of said first downstream probe comprises at least one nucleotide base in said 5'"'"' end which is mismatched with respect to the template sequence to which it hybridizes, and wherein said correction step comprises cleaving the mismatched nucleotide to create a new 5'"'"' phosphorylated terminus on said downsteam probe.

31. The method of claim 30, wherein the 5'"'"' end of the second downstream probe is also modified to be ligation incompetent absent correction, and wherein said correction step includes nucleolytic degradation of the 5'"'"' end of said second downstream probe to create a new phosphorylated 5'"'"' terminus, whereby the correction step renders the 5'"'"' ends of both downstream probes ligation competent.

32. The method of claim 31, wherein said modification of the second downstream probe is selected from:

(a) a non-phosphorylated 5'"'"' terminus;

or (b) at least one nucleotide base in the 5'"'"' end which is mismatched with respect to the template sequence to which it hybridizes; and

wherein said correcting step includes cleaving said non-phosphorylated nucleotide or said mismatched nucleotide.

33. The method of claim 31, wherein said correction step further comprises extending the 3'"'"' terminus of both said upstream probes by the addition of one or more nucleotide triphosphates in a template-dependent manner to bring said extended 3'"'"' termini adjacent to said newly created 5'"'"' phosphorylated termini.

34. The method of claim 32, wherein the 5'"'"' end of said first downstream probe further comprises a non-phosphorylated 5'"'"' terminus.

35. The method of claim 33, wherein both said cleaving and extending steps are performed by a template-dependent polymerase having 5'"'"' to 3'"'"' nucleolytic activity.

36. The method of claim 32, wherein the at least one mismatched base of one or both of said downstream probes is positioned at the 5'"'"' terminal nucleotide.

37. The method of claim 32, wherein the at least one mismatched base of one or both of said downstream probes is positioned within 1 to about 5 nucleotides internal of said 5'"'"' terminal nucleotide.

38. The method of claim 36 or 37, wherein said cleaving step includes cleaving a nucleotide adjacent the mismatched nucleotide on its 3'"'"' side.

39. The method of claim 31, wherein determining the extent of correction and ligation comprises separating ligated product from unligated probes and determining the amount of ligated product formed.

40. The method of claim 31, wherein said terminal nucleotide of the downstream probe carries a label, and wherein said cleaving is performed by a template-dependent polymerase having 5'"'"' to 3'"'"' nucleolytic activity, and further wherein said determining the extent of correction and ligation comprises monitoring the release of label from the downstream probe.

41. A composition of matter comprising:

(a) a first set of oligonucleotides comprising a first upstream probe and a first downstream probe, both probes having sequences substantially complementary to portions of a target nucleic acid sequence, the 3'"'"' terminus of the first upstream probe hybridizing proximate to the 5'"'"' terminus of the first downstream probe; and

(b) a second set of oligonucleotides comprising a second upstream probe and a second downstream probe, both probes having sequences substantially complementary to the first downstream probe and first upstream probes, respectively, the 3'"'"' terminus of the second upstream probe being hybridized proximate to the 5'"'"' terminus of the second downstream probe;

wherein the 5'"'"' end of at least one of the first or second downstream probes is modified to be ligation incompetent absent correction and wherein there is a gap between the 3'"'"' terminus of the hybridized upstream probe and the 5'"'"' terminus of the hybridized downstream probe, or the 5'"'"' terminus of the downstream probe overlaps the 3'"'"' terminus of the upstream probe or the downstream probe comprises a non-phosphorylated 5'"'"' terminus.

42. The composition of claim 41, wherein the ligation incompetent 5'"'"' end comprises a non-phosphorylated 5'"'"' terminus.

43. The composition of claim 42, wherein the non-phosphorylated 5'"'"' terminus is selected from the group consisting of hydroxyl, hydryl, and amino.

44. The composition of claim 42, wherein the non-phosphorylated 5'"'"' terminus includes a label selected from the group consisting of fluorescent labels, radioisotopic labels, chemiluminescent labels, chromophore labels and hapten labels.

45. The composition of claim 41, wherein the ligation incompetent 5'"'"' end comprises at least one nucleotide base in said 5'"'"' end which is mismatched with respect to the target sequence to which it hybridizes or with respect to the complementary upstream probe.

46. The composition of claim 45, wherein said at least one mismatched base is positioned at the 5'"'"' terminal nucleotide.

47. The composition of claim 45, further comprising a non-phosphorylated 5'"'"' terminus.

48. The composition of claim 41, wherein both downstream probes have ligation incompetent 5'"'"' ends.

49. A kit comprising in one or more suitable containers:

(a) a set of oligonucleotides comprising an upstream probe and a downstream probe, both probes having sequences substantially complementary to portions of a target nucleic acid sequence, the 3'"'"' terminus of the first upstream probe hybridizing proximate to the 5'"'"' terminus of the first downstream probe, wherein the 5'"'"' end of said downstream probes is modified to be ligation incompetent absent correction and wherein there is a gap between the 3'"'"' terminus of the hybridized upstream probe and the 5'"'"' terminus of the hybridized downstream probe, or the 5'"'"' terminus of the downstream probe overlaps the 3'"'"' terminus of the upstream probe or the downstream probe comprises a non-phosphorylated 5'"'"' terminus;

(b) one or more correcting reagents for correcting the ligation incompetent downstream probe in a target-dependent manner to render the downstream probe ligatable and for rendering the upstream and downstream probes ligation competent wherein said correcting reagents comprise one or more enzymes having cleaving or cleaving and extending activity; and

(c) a ligating reagent for ligating the corrected downstream probe to the upstream probe.

50. The kit of claim 49, wherein said ligation incompetent 5'"'"' end comprises a non-phosphorylated 5'"'"' terminus.

51. The kit of claim 49, wherein the ligation incompetent 5'"'"' end comprises at least one nucleotide base in said 5'"'"' end which is mismatched with respect to the target sequence to which it hybridizes.

52. The kit of claim 51, further comprising a non-phosphorylated 5'"'"' terminus.

53. The kit of claim 51, wherein said correcting reagent comprises an agent having target-dependent 5'"'"' to 3'"'"' nucleolytic activity.

54. The kit of claim 53 wherein said correcting reagent comprises a target-dependent polymerase.

55. The kit of claim 49, further comprising a second set of oligonucleotides comprising a second upstream probe and a second downstream probe, both probes having sequences substantially complementary to the first downstream probe and first upstream probes, respectively, the 3'"'"' terminus of the second upstream probe being hybridized proximate to the 5'"'"' terminus of the second downstream probe.

56. The kit of claim 55, wherein the ligation incompetent 5'"'"' end on at least one of said downstream probes comprises a non-phosphorylated 5'"'"' terminus.

57. The kit of claim 55, wherein the ligation incompetent 5'"'"' end on at least one of said downstream probes comprises at least one nucleotide base in said 5'"'"' end which is mismatched with respect to the target sequence to which it hybridizes.

58. The kit of claim 57, wherein the ligation incompetent 5'"'"' end on said at least one downstream probe further comprises a non-phosphorylated 5'"'"' terminus.

59. The kit of claim 55, wherein said correcting reagent comprises a target-dependent polymerase having target-dependent 5'"'"' to 3'"'"' nucleolytic activity.