Polymorphism detection and separation

• US 7,468,244 B2
• Filed: 09/27/2002
• Issued: 12/23/2008
• Est. Priority Date: 09/27/2001
• Status: Expired due to Fees

• Current Assignee: University of Delaware
• Original Assignee: University of Delaware
• Inventors: Kmiec, Eric B., Rice, Michael C.
• Primary Examiner(s): BAUSCH, SARAE L
• Application Number:
  US10/260,150
• Publication Number:
  US 20030180746A1
• Time in Patent Office: 2,279 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 536/23.1, 536/24.3
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins

Abstract

Methods and compositions for polymorphism detection and separation. The methods are readily multiplexed, can be adapted to a variety of existing detection systems, and permit target amplification without PCR. The methods permit allelic variants selectively to be isolated, with or without contemporaneous detection, and finds particular utility in facilitating the construction of coisogenic cell collections in which the cells differ genotypically by single nucleotide changes targeted to defined loci.

Claims

1. A method of distinguishing the presence of a nonsupercoiled target nucleic acid from the presence of nonsupercoiled target variants within a sample of nucleic acids having a common target query region, the method comprising:

providing a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotides is bound by a recombinase and said second oligonucleotide is free of a recombinase;

adding said first oligonucleotide to the sample to form a mixture;

adding said second oligonucleotide to the mixture to form a double D loop;

deproteinizing the double D loop; and

distinguishing the double D loops that are stable to deproteinization, wherein the presence of a stable double D loop distinguishes the presence of target from that of variants;

wherein the first oligonucleotide comprises a complementarity region that is perfectly complementary to a first strand of the target across the entirety of the target query region and wherein the second oligonucleotide includes a complementarity region that is perfectly complementary in sequence to a second strand of the target across at least a portion of the target query region, and at least one of said first or second oligonucleotide complementarity regions is imperfectly complementary to at least one of a first strand or a second strand of the query region of the target variants.

2. The method of claim 1, wherein said second oligonucleotide comprises base modifications.

3. The method of claim 1, further comprising the antecedent step of binding a recombinase to said first oligonucleotide.

4. The method of claim 3, wherein said recombinase is E. coli RecA or a mutant thereof.

5. The method of claim 1, wherein said first oligonucleotide complementarity region is no more than 100 nucleotides in length.

6. The method of claim 5, wherein said first oligonucleotide complementarity region is no more than 50 nucleotides in length.

7. The method of claim 1, wherein said first oligonucleotide is no more than 100 nucleotides in length.

8. The method of claim 7, wherein said first oligonucleotide is no more than 50 nucleotides in length.

9. The method of claim 2, wherein said second oligonucleotide comprises base modifications selected from the group consisting of:

LNA bases, PNA bases, RNA bases, and 2′

-OMe bases.

10. The method of claim 9, wherein said second oligonucleotide includes at least 30% modified bases.

11. The method of claim 10, wherein said second oligonucleotide includes at least 50% modified bases.

12. The method of claim 11, wherein said second oligonucleotide includes at least 75% modified bases.

13. The method of claim 1, wherein said second oligonucleotide complementarity region is no more than 50 nucleotides in length.

14. The method of claim 13, wherein said second oligonucleotide complementarity region is no more than 25 nucleotides in length.

15. The method of claim 14, wherein said second oligonucleotide complementarity region is no more than 16 nucleotides in length.

16. The method of claim 1, wherein said second oligonucleotide is no more than 50 nucleotides in length.

17. The method of claim 16, wherein said second oligonucleotide is no more than 25 nucleotides in length.

18. The method of claim 17, wherein said second oligonucleotide is no more than 20 nucleotides in length.

19. The method of claim 1, wherein said first and second oligonucleotide complementarity regions overlap by no more than 25 nucleotides.

20. The method of claim 19, wherein said first and second oligonucleotide complementarity regions overlap by no more than 15 nucleotides.

21. The method of claim 1, wherein at least one of the first and second oligonucleotides includes at least one detectable label.

22. The method of claim 21, wherein said at least one label is selected from the group consisting of:

a radionuclide, a fluorophore, a fluorescence resonance energy transfer tandem fluorophore, a fluorescence resonance energy transfer donor, a fluorescence resonance energy transfer acceptor, a mass tag, an enzyme, a genotypic label, or a hapten.

23. The method of claim 22, wherein said at least one label is a fluorophore.

24. The method of claim 22, wherein said at least one label is a genotypic label.

25. The method of claim 1, wherein said formation of a double D loop is performed at a temperature of at least 37°

C.

26. The method of claim 25, wherein said formation of a double D loop is performed at a temperature of at least 45°

C.

27. The method of claim 26, wherein said formation of a double D loop is performed at a temperature of at least 50°

C.

28. The method of claim 27, wherein said formation of a double D loop at a temperature of at least 55°

C.

29. The method of claim 1, wherein the deproteinizing step is performed at a temperature of at least 37°

C.

30. The method of claim 29, wherein said deproteinizing step is performed for no more than about 10 minutes.

31. The method of claim 1, wherein said double D loops are stable for a time following deproteinization sufficient to permit detectable separation of said target from said variants.

32. The method of claim 31, wherein said separation is electrophoretic separation.

33. The method of claim 1, wherein said double D loops are stable for at least 2 hours at 4°

C. following deproteinization.

34. The method of claim 33, wherein said double D loops are stable for at least 4 hours at 4°

C. following deproteinization.

35. The method of claim 1, wherein said double D loops are stable for at least 30 minutes at 37°

C. following deproteinization.

36. The method of claim 1, wherein said nonsupercoiled double-stranded target is linear duplex DNA.

37. The method of claim 1, wherein said nonsupercoiled double-stranded target is a covalently closed circle.

38. The method of claim 1, wherein said nonsupercoiled double-stranded target is an artificial chromosome.

39. The method of claim 38, wherein the target query region within said artificial chromosome is flanked by recognition sites for a site-specific recombinase.

40. The method of claim 1, wherein the nucleic acids of said sample are pooled from a plurality of individuals.

41. The method of claim 1, wherein the nucleic acids of said sample are from a single individual.

42. The method of claim 1, wherein said nucleic acid sample includes at least one variant that differs from said target by no more than one nucleotide in said query region.

43. The method of claim 40, wherein said nucleic acid sample includes at least one variant that differs from said target by no more than one nucleotide in the target query region.

44. The method of claim 43, wherein said at least one variant is a naturally-occurring variant of said target.

45. The method of claim 42, wherein said at least one variant is a recombinantly-engineered variant of said target.

46. The method of claim 25, further comprising the step, after deproteinizing and before distinguishing said double D loops, of separating the nucleic acids that have double D loops from nucleic acids lacking double D loops.

47. The method of claim 46, wherein at least one of said first and second oligonucleotides includes a capture moiety.

48. The method of claim 47, wherein said capture moiety is biotin.

49. The method of claim 1, further comprising the step, after double D loop formation, of extending by polymerase either or both of the first or second oligonucleotides.

50. The method of claim 49, wherein said extension is a single base extension.

51. The method of claim 49, wherein said extension amplifies at least a portion of the target region.

52. The method of claim 51, wherein said amplification is isothermal.

53. The method of claim 1, further comprising the step of quantifying the absolute or relative abundance of target.

54. A method of distinguishably detecting the presence of a plurality of nonsupercoiled targets within a sample of nucleic acids, the method comprising:

for each of the plurality of targets desired to be detected, providing a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide is bound by a recombinase and said second oligonucleotide is free of a recombinase;

wherein said first oligonucleotide includes a complementarity region that is perfectly complementary in sequence to a first strand of its respective target across the entirety of the target query region, and said second oligonucleotide includes a complementarity region that is perfectly complementary in sequence to a second strand of the same target across at least a portion of the target query region and at least one of said oligonucleotide regions is imperfectly complementary in sequence to respective first and second strands of the query region of each of the other targets desired discriminably to be detected;

adding said first oligonucleotide to the sample to form a mixture;

adding said second oligonucleotide to the mixture to form a double D loop;

deproteinizing the double D loop to form at least one deproteinization-stable double D loop; and

distinguishably detecting at least one deproteinization-stable double D loop formed in the target query region, each target double D loop being distinguishably detectable from all others of the double D loops formed in said sample.

55. The method of claim 54, wherein said second oligonucleotide comprises base modifications, and wherein at least one of said oligonucleotides is distinguishable from the first and second oligonucleotides used to detect each of the others of the plurality of targets desired to be detected.

56. The method of claim 54, further comprising quantifying the relative abundance of each of said targets.

57. The method of claim 54, wherein at least 10 targets are discriminably detected.

58. The method of claim 57, wherein at least 50 targets are discriminably detected.

59. The method of claim 58, wherein at least 100 targets are discriminably detected.

60. The method of claim 54, wherein said plurality of targets are discriminably detected concurrently.

61. The method of claim 60, wherein said targets are detected by microarray hybridization.

62. A method of separating a nonsupercoiled double stranded nucleic acid target from other nonsupercoiled nucleic acids present within a sample of nucleic acids, the method comprising:

providing a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide is bound by a recombinase and said second oligonucleotides is free of a recombinase;

wherein said first oligonucleotide includes a complementarity region that is perfectly complementary in sequence to a first strand of the target across the entirety of the target query region, and said second oligonucleotide includes a complementarity region that is perfectly complementary in sequence to a second strand of the target across at least a portion of the target query region, and at least one of said first or second oligonucleotide complementarity regions is imperfectly complementary to the respective first and second strands of the query region of each of said other nonsupercoiled nucleic acids;

adding said first oligonucleotide to the sample to form a mixture;

adding said second oligonucleotide to the mixture to form a double D loop;

deproteinizing the double D loop to form at least one deproteinization-stable double D loop; and

separating nucleic acids having at least one deproteinization-stable double D loop in the query region of the target, from other nucleic acids present within said sample.

63. The method of claim 62, wherein said second oligonucleotide comprises base modifications.

64. The method of claim 62, wherein at least one of the first and second oligonucleotides includes a capture moiety, and said nucleic acids having deproteinization-stable double D loops are separated from other nucleic acids present within said sample by capture of said moiety.

65. The method of claim 64, wherein said moiety is captured to a solid substrate.

66. The method of claim 65, wherein said solid substrate is a surface of a bead.

67. A method of distinguishing the presence of a supercoiled target nucleic acid from the presence of supercoiled target variants within a sample of nucleic acids, the variants differing from the target by as few as one nucleotide within a common target query region, the method comprising:

providing a first oligonucleotide and a second oligonucleotide, wherein said first oligonucleotide is bound by a recombinase and wherein said second oligonucleotide is free of a recombinase;

wherein the first oligonucleotide includes a complementarity region that is perfectly complementary in sequence to a first strand of the target across the entirety of the target query region, said region being imperfectly complementary to a first strand of the query region of each of said target variants, wherein the first oligonucleotide mediates formation of at least one deproteinization-stable double D loop in the query region of the target;

wherein said second oligonucleotide includes a complementarity region that is perfectly complementary in sequence to a second strand of the target across at least a portion of the target query region;

adding said first oligonucleotide to the sample to form a mixture;

adding said second oligonucleotide to the mixture to form a double D loop;

deproteinizing the double D loop; and

distinguishing the double D loops that are stable to deproteinization, wherein the presence of a stable double D loop distinguishes the presence of target from that of variants.

68. The method of 67, wherein at least one of said first and second oligonucleotide complementarity regions is imperfectly complementary to respective strand of the query region of each of said target variants, and wherein said second oligonucleotide comprises base modifications and wherein said second oligonucleotide is distinguishably detectable.

69. The method of claim 68, further comprising:

providing a third oligonucleotide, wherein said third oligonucleotide is free of a recombinase and comprises base modifications;

adding said third oligonucleotide to the double D loop;

wherein said third oligonucleotide includes a complementarity region that is perfectly complementary in sequence to at least a portion of the second strand of the query region of a target variant as to which the target is desired to be discriminated, and wherein said third oligonucleotide complementary region is imperfectly complementary in sequence to the complementarity region of said first oligonucleotide.