Cleaved amplified RFLP detection methods

• US 6,004,783 A
• Filed: 09/18/1996
• Issued: 12/21/1999
• Est. Priority Date: 03/18/1994
• Status: Expired due to Fees

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.), Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Preuss, Daphne, Davis, Ronald W., Ausubel, Frederick
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US08/715,484
• Time in Patent Office: 1,189 Days
• Field of Search: 435/4, 435/6, 435/91.2, 536/23.1, 536/24.3, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The invention features methods for detecting polymorphic restriction sites in nucleic acids and kits for carrying out these methods.

Claims

1. A method for identifying a polymorphic restriction site in a nucleic acid, said method comprising the steps of:

(a) digesting DNA isolated from a first sample of an organism with a first restriction endonuclease to generate a first reaction product;

(b) ligating to each of the ends of said first reaction product a first adaptor to generate a second reaction product;

(c) digesting said second reaction product with a second restriction endonuclease to generate a third reaction product;

(d) ligating to each of the ends of said third reaction product a second adaptor to generate a fourth reaction product;

(e) amplifying said fourth reaction product to generate a fifth reaction product by PCR using a first primer complementary to said first adaptor and a second primer complementary to said second adaptor, said second primer being tagged with a first member of a specific binding pair;

(f) in a separate set of reactions, digesting DNA isolated from a second sample from said organism with said first restriction endonuclease to generate a sixth reaction product;

(g) ligating to each of the ends of said sixth reaction product a third adaptor to generate a seventh reaction product;

(h) digesting said seventh reaction product with said second restriction endonuclease to generate an eighth reaction product;

(i) denaturing said fifth reaction product to generate a ninth reaction product and denaturing said eighth reaction product to generate a tenth reaction product;

(j) combining said ninth and tenth reaction products under conditions allowing hybridization to generate an eleventh reaction product;

(k) contacting said eleventh reaction product with the second member of said specific binding pair, said second member being immobilized on a solid support;

(l) recovering any of said eleventh reaction product captured on said solid support to generate a twelfth reaction product; and

(m) amplifying said twelfth reaction product by PCR using a primer complementary to said third adaptor, an amplified product being an indication of a polymorphic restriction site that is recognized by said second restriction endonuclease.

2. The method of claim 1, wherein said specific binding pair is avidin and biotin.

51. The method of claim 1, wherein said DNA is obtained from a plant.

52. The method of claim 1, wherein said DNA is obtained from a human.

53. The method of claim 1, wherein said DNA is obtained from a bacterium.

60. The method of claim 53, wherein said method is used for bacterial typing.

3. A kit for identifying a polymoiphic restriction site in a nucleic acid, said kit comprising:

(a) a first DNA adaptor, a second DNA adaptor, and a third DNA adaptor, said first and third DNA adaptors comprising regions complementary to the ends generated by a first restriction endonuclease ends but differing in overall sequence and said second DNA adaptor comprising a region complementary to the ends generated by a second restriction endonuclease, said second restriction endonuclease site corresponding to said polymorphic restriction site; and

(b) a first primer, a second primer, and a third primer, said first primer hybridizing to said first DNA adaptor, said second primer hybridizing to said second DNA adaptor and being tagged with a first member of a specific binding pair, and said third primer hybridizing to said third DNA adaptor.

4. The kit of claim 3, wherein said kit further comprises the second member of said specific binding pair immobilized on a solid support.

5. A method for detecting the presence or absence of a polymorphic restriction site in a nucleic acid, said method comprising the steps of:

(a) amplifying said nucleic acid by PCR using a first and a second primer flanking said polymorphic restriction site, said first primer being tagged with a detectable label, wherein said amplifying generates a PCR product comprising a first strand tagged with said detectable label and an unlabeled second strand;

(b) treating said PCR product with a restriction endonuclease that recognizes said polymorphic restriction site to generate a digestion product;

(c) denaturing said digestion product to generate a denatured product;

(d) contacting said denatured product with a first probe, said first probe comprising a sequence that hybridizes to a first sequence in said first strand, said first sequence being between said polymorphic restriction site and the sequence in said first strand that is complementary to said second primer, said first probe being immobilized on a first binding element;

(e) monitoring said first binding element for the presence of said detectable label, wherein detection of said detectable label on said first binding element indicates the absence of said polymorphic restriction site in said nucleic acid, and a failure to detect said detectable label on said first binding element indicates the presence of said polymorphic restriction site in said nucleic acid.

6. The method of claim 5, further comprising the steps of:

(a) contacting said denatured product with a second, a third, or a fourth probe, said second probe comprising a sequence that hybridizes to a second sequence in said first strand, said second sequence being between said polymorphic restriction site and the sequence in said first strand that is identical to said first primer, said second probe being immobilized on a second binding element;

said third probe comprising a sequence that hybridizes to a third sequence in said second strand, said third sequence being between said polymorphic restriction site and the sequence in said second strand that is identical to the sequence of said second primer, said third probe being immobilized on a third binding element;

said fourth probe comprising a sequence that hybridizes to a fourth sequence in said second strand, said fourth sequence being between said polymorphic restriction site and the sequence in said second strand that is complementary to said first primer, said fourth probe being immobilized on a fourth binding element; and

(b) monitoring said second, third, or fourth binding element for the presence of said detectable label.

7. The method of claim 5, wherein said first binding element is a region on a solid support.

8. The method of claim 7, wherein said solid support is glass.

9. The method of claim 7, wherein said solid support is a microchip.

10. The method of claim 6, wherein said first, second, third, and fourth binding elements are each distinct regions on a solid support.

11. The method of claim 10, wherein said solid support is glass.

12. The method of claim 10, wherein said solid support is a microchip.

54. The method of claim 5, wherein said nucleic acid is obtained from a plant.

55. The method of claim 5, wherein said nucleic acid is obtained from a human.

56. The method of claim 5, wherein said nucleic acid is obtained from a bacterium.

61. The method of claim 56, wherein said method is used for bacterial typing.

13. A method for detecting the presence or absence of a polymorphic restriction site in a nucleic acid, said method comprising the steps of:

(a) amplifying said nucleic acid by PCR using a first and a second primer flanking said polymorphic restriction site, said first primer being tagged with a first detectable label, said second primer being tagged with a second detectable label, wherein said amplifying generates a PCR product comprising a first strand tagged with said first detectable label and a second strand tagged with said second detectable label;

(b) treating said PCR product with a restriction endonuclease that recognizes said polymorphic restriction site to generate a digestion product;

(c) denaturing said digestion product to generate a denatured product;

(d) contacting said denatured product with a first and a second probe, said first probe comprising a sequence that hybridizes to a first sequence in said first strand, said first sequence being between said polymorphic restriction site and the sequence in said first strand that is complementary to said second primer, said first probe being immobilized on a first binding element;

said second probe comprising a sequence that hybridizes to a second sequence in said second strand, said second sequence being between said polymorphic restriction site and the sequence in said second strand that is complementary to said first primer, said second probe being immobilized on a second binding element;

(e) monitoring said first binding element for the presence of said first detectable label and monitoring said second binding element for the presence of said second detectable label, wherein detection of said first detectable label on said first binding element and detection of said second detectable label on said second binding element indicates the absence of said polymorphic restriction site in said nucleic acid, and a failure to detect said first detectable label on said first binding element and a failure to detect said second detectable label on said second binding element indicates the presence of said polymorphic restriction site in said nucleic acid.

14. The method of claim 13, further comprising the steps of:

(a) contacting said denatured product with a third or a fourth probe, said third probe comprising a sequence that hybridizes to a third sequence in said first strand, said third sequence being between said polymorphic restriction site and the sequence in said first strand that is identical to the sequence of said first primer, said third probe being immobilized on a third binding element;

said fourth probe comprising a sequence that hybridizes to a fourth sequence in said second strand, said fourth sequence being between said polymorphic restriction site and the sequence in said second strand that is identical to the sequence of said second primer, said fourth probe being immobilized on a fourth binding element; and

(b) monitoring said third or fourth binding element for the presence of said first or second detectable label.

15. The method of claim 13, wherein said first and said second detectable labels are identical.

16. The method of claim 13, wherein said first and second binding elements are each distinct regions on a solid support.

17. The method of claim 16, wherein said solid support is glass.

18. The method of claim 16, wherein said solid support is a microchip.

19. The method of claim 14, wherein said first, second, third, and fourth binding elements are each distinct regions on a solid support.

20. The method of claim 19, wherein said solid support is glass.

21. The method of claim 19, wherein said solid support is a microchip.

57. The method of claim 13, wherein said nucleic acid is obtained from a plant.

58. The method of claim 13, wherein said nucleic acid is obtained from a human.

59. The method of claim 13, wherein said nucleic acid is obtained from a bacterium.

62. The method of claim 59, wherein said method is used for bacterial typing.

22. A method for determining whether an organism is homozygous for a polymorphic restriction site, is heterozygous for said polymorphic restriction site, or lacks said polymorphic restriction site, said method comprising the steps of:

(a) carrying out PCR on a sample comprising nucleic acid molecules from the organism using a first and a second primer flanking said polymorphic restriction site, wherein said amplifying generates a PCR product comprising a first strand comprising a sequence that is identical to the sequence of said first primer and a second strand comprising a sequence that is identical to the sequence of said second primer;

(b) treating said PCR product with a restriction endonuclease that recognizes said polymorphic restriction site to generate a digestion product;

(c) denaturing said digestion product to generate a denatured product;

(d) contacting said denatured product with an oligonucleotide to generate a first reaction product, said oligonucleotide comprising a 3'"'"' portion that hybridizes to a first region in said first strand, said first region being between said polymorphic restriction site and a sequence in said first strand that is identical to the sequence of said first primer, said oligonucleotide being blocked so that it cannot serve as a primer for DNA polymerase, said oligonuclcotidc comprising a 5'"'"' portion that does not hybridize to said first strand;

(e) treating said first reaction product with a DNA polymerase to extend any unblocked, primed 3'"'"' end in said first reaction product to generate a second reaction product;

(f) amplifying said second reaction product by PCR using a third primer, said third primer having a sequence identical to said first primer and being tagged with a first detectable label, and a fourth primer that hybridizes to a sequence that is complementary to said 5'"'"' portion of said oligonucleotide to generate a second PCR product, said fourth primer being tagged with a second detectable label;

(g) denaturing said second PCR product to generate a second denatured product;

(h) contacting said second denatured product with a first and a second probe, said first probe comprising a sequence that hybridizes to a first sequence in said second strand, said first sequence being between said polymorphic restriction site and the sequence in said second strand that is complementary to said first primer, said first probe being immobilized on a first binding element;

said second probe comprising a sequence that hybridizes to a second sequence in said first strand, said second sequence being between said polymorphic restriction site and the sequence in said first strand that is complementary to said second primer, said second probe being immobilized on a second binding clement;

(i) monitoring said first binding element for the presence of said second detectable label and monitoring said second binding element for the presence of said first detectable label, wherein detection of said second detectable label on said first binding element and detection of said first detectable label on said second binding element indicates that the organism is heterozygous for said polymorphic restriction site, detection of said second detectable label on said first binding element and a failure to detect said first detectable label on said second binding element indicates that the organism is a homozygote comprising said polymorphic restriction site, and detection of said first detectable label on said second binding element and a failure to detect said second detectable label on said first binding element indicates that the organism is a homozygote lacking said polymorphic restriction site.

23. The method of claim 22, further comprising the steps of:

(a) contacting said second denatured product with a third or a fourth probe, said third probe comprising a sequence that hybridizes to a third sequence in said first strand, said third sequence being between said polymorphic restriction site and the sequence in said first strand that is identical to the sequence of said first primer, said third probe being immobilized on a third binding element;

said fourth probe comprising a sequence that hybridizes to a fourth sequence in said second strand, said fourth sequence being between said polymorphic restriction site and the sequence in said second strand that is identical to the sequence of said second primer, said fourth probe being immobilized on a fourth binding element; and

(b) monitoring said third or fourth binding element for the presence of said first or second detectable label.

24. The method of claim 22, wherein said first and said second detectable labels are identical.

25. The method of claim 22, wherein said first and second binding elements are each distinct regions on a solid support.

26. The method of claim 25, wherein said solid support is glass.

27. The method of claim 25, wherein said solid support is a microchip.

28. The method of claim 23, wherein said first, second, third, and fourth binding elements are each distinct regions on a solid support.

29. The method of claim 28, wherein said solid support is glass.

30. The method of claim 28, wherein said solid support is a microchip.

63. The method of claim 22, wherein said nucleic acid is obtained from a plant.

64. The method of claim 22, wherein said nucleic acid is obtained from a human.

65. The method of claim 22, wherein said nucleic acid is obtained from a bacterium.

66. The method of claim 65, wherein said method is used for bacterial typing.

31. A kit for detecting the presence or absence of a polymorphic restriction site in a nucleic acid, said kit comprising:

a first and a second primer flanking said polymorphic restriction site, said first primer being tagged with a detectable label, wherein amplifying said nucleic acid by PCR with said first and second primers generates a PCR product comprising a first strand tagged with said detectable label and a second strand; and

a first probe that comprises a sequence that hybridizes to a first sequence in said first strand, said first sequence being between said polymorphic restriction site and the sequence in said first strand that is complementary to said second primer, and said first probe is immobilized on a first binding element.

32. The kit of claim 31, further comprising a second, third, or fourth probe, said second probe comprising a sequence that hybridizes to a second sequence in said first strand, said second sequence being between said polymorphic restriction site and the sequence in said first strand that is identical to the sequence of said first primer, said second probe being immobilized on a second binding element;

said third probe comprising a sequence that hybridizes to a third sequence in said second strand, said third sequence being between said polymorphic restriction site and the sequence in said second strand that is identical to the sequence of said second primer, said third probe being immobilized on a third binding element; and

said fourth probe comprising a sequence that hybridizes to a fourth sequence in said second strand, said fourth sequence being between said polymorphic restriction site and the sequence in said second strand that is complementary to said first primer, said fourth probe being immobilized on a fourth binding element.

33. The kit of claim 31, wherein said first binding element is a region on a solid support.

34. The kit of claim 32, wherein said solid support is glass.

35. The kit of claim 32, wherein said solid support is a microchip.

36. The kit of claim 31, wherein said first, second, third, and fourth binding elements are each distinct regions on a solid support.

37. The kit of claim 36, wherein said solid support is glass.

38. The kit of claim 36, wherein said solid support is a microchip.

39. The kit of claim 31, wherein said second primer comprises a second detectable label and said kit further comprises a second probe comprising a sequence that hybridizes to a second sequence in said second strand, said second sequence being between said polymorphic restriction site and the sequence in said second strand that is complementary to said first primer, and said second probe being immobilized on a second binding element.

40. The kit of claim 39, further comprising a third or a fourth probe, said third probe comprising a sequence that hybridizes to a third sequence in said first strand, said third sequence being between said polymorphic restriction site and the sequence in said first strand that is identical to the sequence of said first primer, said third probe being immobilized on a third binding element;

said fourth probe comprising a sequence that hybridizes to a fourth sequence in said second strand, said fourth sequence being between said polymorphic restriction site and the sequence in said second strand that is identical to the sequence of said second primer, and said fourth probe being immobilized on a fourth binding element.

41. The kit of claim 31, further comprising a second probe comprising a sequence that hybridizes to a second sequence in said first strand, said second sequence being between said polymorphic restriction site and the sequence in said first strand that is identical to said first primer, and said second probe being immobilized on a second binding element.

42. A kit for detecting the presence or absence of a polymorphic restriction site in a nucleic acid, said kit comprising:

a first and a second PCR primer flanking said polymorphic restriction site, said first primer being tagged with a first detectable label, wherein amplifying said nucleic acid by PCR using said first and second primers generates a PCR product comprising a first strand tagged with said first detectable label and a second strand;

an oligonucleotide comprising a 3'"'"' portion that hybridizes to a first region in said first strand, said first region flanking said polymorphic restriction site on the side of said polymorphic restriction site comprising a sequence that is identical to the sequence of said first primer, said oligonucleotide being blocked so that it cannot serve as a primer for DNA polymerase, said oligonucleotide comprising a 5'"'"' portion that does not hybridize to a second region in said first strand, said second region flanking said polymorphic restriction site on the side of said polymorphic restriction site comprising a sequence that is complementary to said second primer;

a third primer that hybridizes to a sequence that is complementary to said 5'"'"' portion of said oligonucleotide, said third primer being tagged with a second detectable label;

a first and a second probe, said first probe comprising a sequence that hybridizes to a first sequence in said second strand, said first sequence being between said polymorphic restriction site and the sequence in said second strand that is complementary to said first primer, said first probe being immobilized on a first binding element;

said second probe comprising a sequence that hybridizes to a second sequence in said first strand, said second sequence being between said polymorphic restriction site and the sequence in said first strand that is complementary to said second primer, and said second probe being immobilized on a second binding element.

43. The kit of claim 42, further comprising a third or a fourth probe, said third probe comprising a sequence that hybridizes to a third sequence in said first strand, said third sequence being between said polymorphic restriction site and the sequence in said first strand that is identical to the sequence of said first primer, said third probe being immobilized on a third binding element;

said fourth probe comprising a sequence that hybridizes to a fourth sequence in said second strand, said fourth sequence being between said polymorphic restriction site and the sequence in said second strand that is identical to the sequence of said second primer, and said fourth probe being immobilized on a fourth binding element.

44. The kit of claim 42, wherein said first and said second detectable labels are identical.

45. The kit of claim 42, wherein said first and second binding elements are each distinct regions on a solid support.

46. The kit of claim 45, wherein said solid support is glass.

47. The kit of claim 45, wherein said solid support is a microchip.

48. The kit of claim 43, wherein said one or more sets of said first, second, third, and fourth binding elements are each distinct regions on a solid support.

49. The kit of claim 48, wherein said solid support is glass.

50. The kit of claim 48, wherein said solid support is a microchip.