Nucleic acid enrichment

• US 20050037356A1
• Filed: 11/20/2002
• Published: 02/17/2005
• Est. Priority Date: 11/20/2001
• Status: Active Grant

• Current Assignee: Olink AB (Thermo Fisher Scientific Incorporated)
• Original Assignee: Olink AB (Thermo Fisher Scientific Incorporated)
• Inventors: Landegren, Ulf, Gullberg, Mats
• Granted Patent: US 8,053,188 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 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/137 incorporating/modifying moieties to eliminate restriction sites
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]

Abstract

This invention relates to methods, reagents and kits for enriching nucleic acid sequences. More particularly, the present invention relates to methods, reagents and kits for sample preparation including sample modification, sample enrichment and amplification

Claims

1. of enriching a preselected nucleic acid segment from a mixture of nucleic acid sequences, the preselected nucleic acid sequence encompassing a specific variant at a given position, the method comprising the steps of:

(a) providing a nucleic acid mixture of sequences which includes the preselected nucleic acid segment to be enriched;

(b) cleaving the nucleic acid sequences in the mixture to provide a nucleic acid fragment comprising the preselected nucleic acid segment;

(c) providing a template oligonucleotide, one end of which hybridises to a sequence of the segment at or close to the variant position, and the other end of which hybridises to the end of a protecting sequence;

(d) hybridising the template to the nucleic acid segment and to the protecting sequence such that the variant position and the end of the protecting sequence are brought into proximity of each other;

(e) joining the end of the protecting sequence to the nucleic acid segment to form a ligated product, which ligated product is protected from degradation; and

(f) enriching for the ligated sequence.

2. A method according to claim 1, step (b), in which cleavage generates a variant position located either at or close to one of the ends.

3. A method according to claim 1, step (f), in which the un-ligated product is degraded.

4. A method according to claim 1, step (f) in which the ligated product is amplified.

5. A method according to claim 1, step (f), in which un-ligated product is degraded and ligated product is amplified.

6. A method according to claim 1, step (b), in which the variant position is located at one end of the nucleic acid sequence and a defined nucleic acid sequence is located at the opposite end.

7. A method according to claim 1, in which the cleavage generates a variant position located at or downstream of the 5′

end of the segment.

8. A method according to claim 3, in which degradation is effected with an exonuclease enzyme.

9. A method as claimed in claim 1, in which the hybridisation of the template generates a structure that is a substrate for cleavage by a structure specific enzyme.

10. A method according to claim 9, in which the structure specific enzyme is selected from the group including any one or more;

Native or recombinant Fen nuclease,

Native or recombinant Mja nuclease

Recombinant polymerase from Thermus aquatiqus,

Native or recombinant polymerase from Thermus thermophilus, and

Native or recombinant polymerase from Thermus flavus.

11. A method according to claim 9, in which the structure specific enzyme is native polymerase from Thermus aquaticus.

12. A method according to claim 1, in which the protecting sequence is the opposite end of the nucleic acid segment, and in which the ligating step (1e) circularises the nucleic acid segment.

13. A method according to claim 1, in which the nucleic acid segment comprises DNA.

14. A method according to claim 1, in which the nucleic acid segment comprises RNA.

15. A method according to claim 1, in which the amplifying step comprises rolling circle amplification.

16. A method according to claim 1, in which the protecting sequence is a nucleic acid adapter with a protected end.

17. A method according to claim 1, in which, cleavage of the nucleic acid sample in step (lb) uses at least one adapter and at least one restriction enzyme.

18. A method according to claim 17 in which, the one or more adapters hybridises to the nucleic acid sample such that, upon cleavage, the variant position is located at one end of the segment.

19. A method according to claim 1, in which there is a gap between the hybridised 3′

end and the hybridised upstream sequence of the nucleic acid segment.

20. A method according to claim 19 in which, one of the sequences is extended to fill the gap there between.

21. A method according to claim 19, in which one of the sequences is extended with a polymerase.

22. A method according to claim 19, in which the sequence is extended with an enzyme selected from the group including any or more;

Native or recombinant polymerase from Thermus aquatiqus,

Native or recombinant polymerase from Thermus thermophilus, and

Native or recombinant polymerase from Thermus flavus.

23. A method according to claim 19, in which one of the sequences is extended with Klenow.

24. A method according to claim 19 in which, the gap is closed in with an oligonucleotide.

25. A method according to claim 24, in which the oligonucleotide inserted into the gap is labelled with an affinity tag.

26. A method according to claim 24, in which the oligonucleotide is labelled with biotin.

27. A method as claimed in claim 1, which includes an initial step of denaturing the nucleic acid when the nucleic acid is at least partially double stranded.

47. A method according to claim 1, in which the specific variant is a polymorphism, wherein the process includes a further step of variant scorng.

48. A method according to claim 1, in which the specific variant is a single nucleotide variant, wherein the process includes a further step of single nucleotide variant scoring.

49. A method according to claim 1, in which the specific variant is a deletion variant, wherein the process includes a further step of deletion variant scoring.

50. A method according to claim 1, in which the specific variant is an insertion variant wherein the process includes a further step of insertion variant scoring.

51. A method according to claim 1, in which the specific variant is a sequence variation wherein the process includes a further step of sequence variation scoring.

52. A method according to claim 1, in which the specific variant is a sequence length variation, wherein the process includes a further step of sequence length variation scoring.

62. A method according to claim 1, in which the protecting sequence is the opposite end of the nucleic acid segment, and in which the ligating step (1e) circularises the nucleic acid segment.

63. A method according to claim 1, in which the nucleic acid segment comprises DNA.

64. A method according to claim 1, in which the nucleic acid segment comprises RNA.

65. A method according to claim 1, in which the amplifying step comprises rolling circle amplification.

67. A method according to claim 1, in which the protecting sequence is a nucleic acid adapter with a protected end.

68. A method according to claim 1, in which, cleavage of the nucleic acid sample in step (53b) uses at least one adapter and at least one restriction enzyme.

69. A method according to claim 67 in which, the one or more adapters hybridises to the nucleic acid sample such that, upon cleavage, the variant position is located at one end of the segment.

70. A method according to claim 1, in which there is a gap between the hybridised 3′

end and the hybridised upstream sequence of the nucleic acid segment.

71. A method according to claim 69 in which, one of the sequences is extended to fill the gap there between.

72. A method according to claim 69, in which one of the sequences is extended with a polymerase.

73. A method according to claim 70, in which the sequence is extended with an enzyme selected from the group including any or more;

Native or recombinant polymerase from Thermus aquatiqus,

Native or recombinant polymerase from Thermus thermophilus, and

Native or recombinant polymerase from Thermus flavus.

74. A method according to claim 70, in which one of the sequences is extended with Klenow.

75. A method according to claim 70 in which, the gap is closed in with an oligonucleotide.

76. A method according to claim 75, in which the oligonucleotide inserted into the gap is labelled with an affinity tag.

77. A method according to claim 75, in which the oligonucleotide is labelled with biotin.

78. A method as claimed in claim 1, which includes an initial step of denaturing the nucleic acid when the nucleic acid is at least partially double stranded.

28. A method of analysing a target nucleic acid sequence for the presence of a mutation at a given variant position comprising the steps of:

a) providing a probe for the target sequence, the probe having a 3′

end which is complementary to part of the target sequence, and having a sequence downstream of the 5′

end which is complementary to an adjacent part of the target sequence when the target sequence includes the mutation;

b) hybridising the probe with the target sequence, forming a non-hybridising region at the 5′

end of the probe;

c) cleavage of the non hybridising region of the probe by a structure specific enzyme;

d) circularisation of the probe;

e) removal of uncircularised probe;

f) optionally amplifying the circularised probe;

g) and detection of the amplified product.

29. A method according to claim 28, in which the structure specific enzyme is selected from the group including any one or more;

Native or recombinant Fen nuclease,

Native or recombinant Mja nuclease

Recombinant polymerase from Thermus aquatiqus,

Native or recombinant polymerase from Thermus thermophilus, and

Native or recombinant polymerase from Thermus flavus.

30. A method according to claim 28, in which the structure specific enzyme is native Taq polymerase.

31. A method according to claim 28, in which the removal ofuncircularised probes in step (28e) comprises exonuclease treatment.

32. A method according to any claim 28, in which the removal ofuncircularised probes (28e) is by binding to a portion in the 5′

end of the probe that is cleaved off if hybridised to the sample.

33. A method according to claim 28, in which the amplification step (28f) comprises rolling circle amplification.

34. A method according claim 28, in which the detection of the product, step (28g) is by detection of the circularised probe directly or by detection of amplification products thereof.

35. A method according to claim 28, in which a gap is present between the hybridised 3′

end and the hybridised upstream sequence.

36. A method according to claim 28, in which one of the sequences is extended to fill the gap therebetween.

37. A method according to claim 35, in which one of the sequences is extended with a polymerase.

38. A method according to claim 36, in which the sequence is extended with an enzyme selected from the group including any one or more;

Recombinant polymerase from Thermus aquatiqus,

Native or recombinant polymerase from Thermus thermophilus, and

Native or recombinant polymerase from Thermus flavus.

39. A method according to claim 36 in which one of the sequences is extended with native polymerase from Thermus aquaticus.

40. A method according to claim 36, in which one of the sequences is extended with Klenow.

41. A method according to claim 35 in which, the gap is closed in with an oligonucleotide.

42. A method according to claim 35 in which the oligonucleotide inserted into the gap is labelled with an affinity tag.

43. A method according to claim 28, in which the oligonucleotide is labelled with biotin.

44. A method according to claim 28 in which the probe is labelled with a detectable cleavable molecule.

45. A method according to claim 44 in which, the detectable cleavable molecule is fluorescent.

46. A method according to claim 44, in which cleavage of the probe is detected by fluorescence.

53. A method of enriching a preselected nucleic acid segment from a mixture of nucleic acid sequences, the method comprising the steps of:

(a) providing a nucleic acid mixture of sequences which includes the preselected nucleic acid segment to be enriched;

(b) cleaving the nucleic acid sequences in the mixture to provide a nucleic acid fragment comprising the preselected nucleic acid segment;

(c) providing a template oligonucleotide, one end of which hybridises to a sequence of the segment, and the other end of which hybridises to the end of a protecting sequence;

(d) hybridising the template to the nucleic acid segment and to the protecting sequence such that the nucleic acid segment and the protecting sequence are brought into proximity of each other;

(e) joining the end of the protecting sequence to the nucleic acid segment to form a ligated product, which ligated product is protected from degradation; and

(f) enriching for the ligated sequence.

54. A method according to claim 53, step (f), in which the un-ligated product is degraded.

55. A method according to claim 53, step (f) in which the ligated product is amplified.

56. A method according to claim 53, step (f), in which un-ligated product is degraded and ligated product is amplified.

57. A method according to claim 53, step (b), in which a defined nucleic acid sequence is located at least at one of the ends.

58. A method according to claim 53, in which degradation of steps (e) and (f) is effected with an exonuclease enzyme.

59. A method as claimed in claim 53, in which the hybridisation of the template generates a structure that is a substrate for cleavage by a structure specific enzyme.

60. A method according to claim 59, in which the structure specific enzyme is selected from the group including any one or more;

Native or recombinant Fen nuclease,

Native or recombinant Mja nuclease

Recombinant polymerase from Thermus aquatiqus,

Native or recombinant polymerase from Thermus thermophilus, and

Native or recombinant polymerase from Thermus flavus.

61. A method according to claim 59, in which the structure specific enzyme is native polymerase from Thermus aquaticus.

66. A method according to claim 55, in which amplification is performed with native or recombinant polymerase of phage phi29 or related polymerases.

79. A method according to claim 55, in which the amplification product is labelled during amplification.

80. A method as claimed in claim 53, in which the selected segment is subjected to a further step of nucleotide sequencing.

81. A method as claimed in claim 53, in which the specific segment is subjected to a further step of single nucleotide variant scoring.

82. A method as claimed in claim 53, in which the specific segment is subjected to a further step of deletion variant scoring.

83. A method as claimed in claim 53, in which the specific segment is subjected to a further step of insertion variant scoring.

84. A method as claimed in claim 53, in which the specific segment is subjected to a further step of sequence variation scoring.

85. A method as claimed in claim 53, in which the specific segment is subjected to a further step of sequence length variation scoring.

86. A method as claimed in claim 53, in which the specific segment is subjected to a further step of use as hybridisation probe for in situ analysis.

87. A method as claimed in claim 53, in which the specific segment is subjected to a further step of hybridisation to an array.

88. A method as claimed in claim 53, in which the specific segment is subjected to a further step of quantification.