METHOD OF MAKING A PAIRED TAG LIBRARY FOR NUCLEIC ACID SEQUENCING

• US 20090181861A1
• Filed: 01/08/2009
• Published: 07/16/2009
• Est. Priority Date: 01/09/2008
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Li, Bin, Ranade, Swati S., Wang, Yangzhou, Xi, Lei
• Granted Patent: US 8,530,197 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides

Abstract

The present disclosure relates to methods and compositions for making paired tags and paired tag libraries.

Claims

1. A method of forming a paired tag comprising a first tag sequence and a second tag sequence that together comprise at least part of a polynucleotide of interest, said method comprising:

ligating a first end and a second end of a double stranded polynucleotide of interest to an adaptor thereby forming a circular nucleic acid molecule, wherein the circular nucleic acid molecule comprises a first nick between the first end of the double stranded polynucleotide of interest and the adaptor, and a second nick between the second end of the double stranded polynucleotide of interest and the adaptor, wherein the first nick and the second nick are on different strands of the circular nucleic acid molecule than the second nick; and

performing a nick translation reaction wherein at least one nick is translated into the polynucleotide of interest.

2. The method of claim 1, further comprising:

allowing the nick translation reaction to proceed for a specific time; and

terminating the nick translation reaction.

3. The method of claim 1, wherein the first end and the second end of the double stranded polynucleotide of interest lack 5′

phosphate residues.

4. The method of claim 1, further comprising terminating the nick translation reaction before the first nick and the second nick are translated past each other.

5. The method of claim 1, wherein the first nick and the second nick are translated more than 10 bases, further comprising the step of cleaving the circular nucleic acid molecule at the first nick and at the second nick.

6. The method of claim 5, wherein the first nick and the second nick are translated less than 500 bases.

7. The method of claims 6, wherein the first nick and the second nick are translated less than 200 bases.

8. The method of claim 7, wherein the first nick and the second nick are translated less than 100 bases.

9. The method of claim 5, wherein the first nick and the second nick are translated between about 20 bases to about 50 bases.

10. The method of claim 1, wherein at least one nick is translated less than 500 bases.

11. The method of claims 10, wherein at least one nick is translated more than 27 bases and less than 500 bases.

12. The method of claim 11, wherein at least one nick is translated less than 200 bases.

13. The method of claim 1, wherein at least one nick is translated from 28 bases to about 50 bases.

14. The method of claim 1, wherein the circular nucleic acid molecule comprises a first gap between the first end of the double stranded polynucleotide of interest and the adaptor.

15. The method of claim 14, wherein the circular nucleic acid molecule comprises a second gap between the second end of the double stranded polynucleotide of interest and the adaptor.

16. The method of claim 14, wherein the circular nucleic acid sequence comprises a gap that is filled in with nucleotides when the nick translation reaction is performed, thereby resulting in a nick.

17. The method of claim 1, wherein the polynucleotide of interest comprises from 50 to 2500 nucleotides.

18. The method of claim 17, wherein the polynucleotide of interest is subjected to a size selection technique to select for polynucleotides comprising from 50 to 2500 nucleotides prior to the ligating step.

19. The method of claim 1, wherein the adaptor comprises a binding moiety.

20. The method of claim 1, wherein the nick translation reaction is performed using an enzyme selected from the group consisting of E. Coli DNA polymerase I, Taq DNA polymerase, Vent DNA polymerase, Klenow DNA polymerase I, and phi29 DNA polymerase and any combination thereof.

21. The method of claim 1, wherein the nick translation reaction is performed using a DNA polymerase having 5′

to 3′

exonuclease activity.

22. The method of claim 1, wherein the nick translation reaction comprises removal of nucleotides by 5′

to 3′

exonuclease activity.

23. The method of claim 1, wherein the nick translation reaction occurs by strand displacement.

24. The method of claim 1, further comprising cleaving the circular nucleic acid molecule at a first translated nick, thereby forming a linear polynucleotide comprising a first tag sequence and a second tag sequence separated by the adaptor, wherein the first tag sequence and the second tag sequence are derived from the polynucleotide of interest.

25. The method of claim 24, further comprising cleaving the circular nucleic acid molecule at a second translated nick.

26. The method of claim 24, wherein the circular nucleic acid molecule is cleaved by an enzyme selected from the group consisting of S1 nuclease, mung bean nuclease, nuclease P1, nuclease BAL-31, and any combination thereof.

27. The method of claim 24, further comprising amplifying the linear polynucleotide.

28. The method of claim 1, wherein the adaptor comprises a binding moiety that comprises biotin.

29. The method of claim 28, further comprising the step of using streptavidin to purify the paired tag.

30. The method of claim 1, further comprising treating the polynucleotide of interest with an alkaline phosphatase prior to ligation, wherein a 5′

-phosphate is removed from both the first end and the second end of the polynucleotide of interest.

31. The method of claim 30, wherein the alkaline phosphatase is selected from the group consisting of calf intestinal alkaline phosphatase, bacterial alkaline phosphatase, shrimp alkaline phosphatase, and some combination thereof.

32. The method of claim 1, wherein the circular nucleic acid molecule further comprises a second adaptor, wherein the second adaptor is positioned adjacent to the first adaptor.

33. The method of claim 32, wherein one strand of the first adaptor and one strand of the second adaptor lack a 5′

phosphate.

34. The method of claim 1, wherein each step is performed in the order listed in claim 1.

35. A method of forming a released paired tag from a DNA fragment of interest, comprising:

ligating a first adaptor to a first end of a DNA fragment of interest and ligating a second adaptor to a second end of the DNA fragment of interest, thereby producing an adaptor modified fragment,

circularizing the adaptor modified fragment by attaching a third adaptor to the adaptor modified fragment, thereby forming a circular nucleic acid molecule wherein a first nick is present between the third adaptor and the first adaptor and a second nick is present between the second adaptor and the third adaptor, wherein the circular nucleic acid molecule comprises a first strand and a second strand of DNA, and wherein the first nick and the second nick are not present on the same strand of the circular nucleic acid molecule;

performing a nick translation reaction wherein a nick on each strand of the circular nucleic acid molecule is translated into the DNA fragment of interest, and

cleaving the circular nucleic acid molecule at a position at a translated nick, thereby forming a released paired tag.

36. The method of claim 35, further comprising:

allowing the nick translation reaction to proceed for a specific time; and

terminating the nick translation reaction.

37. The method of claim 36, further comprising terminating the nick translation reaction prior to cleaving the polynucleotide strand at each position at a translated nick.

38. The method of claim 35, further comprising attaching primer adaptors to the ends of the paired tag.

39. The method of claim 38, further comprising amplifying the paired tag with primers for the primer adaptors.

40. The method of claim 39, wherein the amplification is a clonal amplification.

41. The method of claim 40, wherein the clonal amplification comprises emulsion polymerase chain reaction (emulsion PCR).

42. The method of claim 40, wherein the clonal amplification comprises bridge polymerase chain reaction (bridge PCR).

43. The method of claim 35, wherein the third adaptor comprises a binding moiety, and further comprising binding the binding moiety to a solid support.

44. The method of claim 42, wherein the solid support is an array.

45. The method of claim 35, further comprising sequencing a portion of the paired tag.

46. A released paired tag library comprising two or more released paired tags, wherein each of the paired tags is prepared by a method comprising:

ligating a first adaptor to a first end of a polynucleotide of interest and ligating a second adaptor to a second end of the polynucleotide of interest, thereby producing an adaptor modified polynucleotide of interest,

circularizing the adaptor modified polynucleotide of interest by attaching a third adaptor to the adaptor modified polynucleotide of interest, thereby forming a circular nucleic acid molecule wherein a first nick is present between the third adaptor and the first adaptor and a second nick is present between the second adaptor and the third adaptor, wherein the circular nucleic acid molecule comprises a first strand and a second strand, and wherein the first nick and the second nick are not present on the same strand of the circular nucleic acid molecule;

performing a nick translation reaction wherein a nick on a strand of the circular nucleic acid molecule is translated into the polynucleotide of interest, and

cleaving the circular nucleic acid molecule at a position at a translated nick, thereby forming a released paired tag.

47. The released paired tag library of claim 46, wherein the polynucleotide of interest is prepared from an organism selected from the group consisting of a plasmid, a virus, a prokaryotic cell, an archaebacterial cell, a bacterial artificial chromosome, a eukaryotic cell, a cell line, a protozoan, a plant, an alga, a bacterium, a fungus, an insect, a reptile, a fish, an amphibian, a bird, and a mammal.

48. The released paired tag library of claim 46, wherein the polynucleotide of interest is subjected to a size selection technique.

49. The released paired tag library of claim 48, wherein the size selection technique is used to select for polynucleotides comprising from 2000 to 3000 nucleotides prior to the ligating step.

50. A released paired tag, said paired tag comprising:

a first tag sequence, wherein the first tag sequence comprises a first end of a polynucleotide of interest, and wherein the first tag sequence is at least 27 nucleotides in length;

a first adaptor covalently bonded to the first tag sequence; and

a second tag sequence covalently bonded to the first adaptor, wherein the second tag sequence comprises a second end of the polynucleotide of interest, and wherein the second tag sequence is at least 27 nucleotides in length.

51. The released paired tag of claim 50, wherein the first adaptor further comprises a binding moiety.

52. The released paired tag of claim 50, wherein the first tag sequence is at least 40 nucleotides in length.

53. The released paired tag of claim 52, wherein the second tag sequence is at least 40 nucleotides in length.

54. A solution comprising:

a circular nucleic acid molecule, the circular nucleic acid molecule comprising;

a double stranded polynucleotide of interest comprising a first end and a second end; and

at least one adaptor, wherein the at least one adaptor is covalently bonded to the first end and the second end of the double stranded polynucleotide of interest, thereby forming a circularized nucleic acid molecule, wherein the circular nucleic acid molecule further comprises a first nick between the first end of the double stranded polynucleotide of interest and the adaptor, and a second nick between the second end of the double stranded polynucleotide of interest and the adaptor, and wherein the first nick is on a different strand of the circular nucleic acid molecule than the second nick or gap; and

a nick translation enzyme.

55. The solution of claim 54, wherein the nick translation enzyme comprises DNA polymerase I.

56. The solution of claim 54, wherein the solution further comprises a nick cleavage enzyme and wherein the nick translation enzyme is heat inactivated.

57. The solution of claim 54, wherein the solution further comprises a second circular nucleic acid molecule, the second circular nucleic acid molecule comprising:

the double stranded polynucleotide of interest comprising the first end and the second end; and

the at least one adaptor, covalently bonded to the first end and the second end of the double stranded polynucleotide of interest, thereby forming a second circularized nucleic acid molecule, wherein the second circular nucleic acid molecule comprises a third nick, wherein the third nick is more than 27 nucleotides away from the location between the first end of the double stranded polynucleotide of interest and the adaptor, and a fourth nick, wherein the fourth nick is more than 27 nucleotides away from the location between the second end of the double stranded polynucleotide of interest and the adaptor, and wherein the third nick is on a different strand of the circular nucleic acid molecule than the fourth nick.

58. The solution of claim 57, wherein the third nick is at least 50 nucleotides away from the location between the first end of the double stranded polynucleotide of interest and the adaptor.

59. The solution of claim 58, wherein the fourth nick is at least 50 nucleotides away from the location between the second end of the double stranded polynucleotide of interest and the adaptor.

60. The solution of claim 59, wherein the solution further comprises a nick cleavage enzyme and wherein the nick translation enzyme is heat inactivated.

61. A method of forming a paired tag comprising a first tag sequence and a second tag sequence that together comprise at least part of a polynucleotide of interest, said method comprising:

ligating a first end of a double stranded polynucleotide of interest to at least one adaptor;

ligating a second end of the double stranded polynucleotide of interest to at least one adaptor;

forming a circular nucleic acid molecule comprising the double stranded polynucleotide of interest, a first nick comprising a first 3′

end on a first strand of the circular nucleic acid molecule and a second nick comprising a second 3′

end on a second strand of the circular nucleic acid molecule, wherein the first nick and the second nick are on different strands of the circular nucleic acid molecule;

extending the first 3′

end of the first strand of the circular nucleic acid molecule into a sequence of the double stranded polynucleotide of interest; and

extending the second 3′

end of the second strand of the circular nucleic acid molecule into a sequence of the double stranded polynucleotide of interest.

62. The method of claim 61, wherein prior to extending the first 3′

end, the first nick is located between the first end of the double stranded polynucleotide of interest and the at least one adaptor and the second nick is located between the second end of the double stranded polynucleotide of interest and the at least one adaptor.

63. The method of claim 62, wherein the first and second ends of the double stranded polynucleotide of interest are connected to a same adaptor.

64. The method of claim 61, wherein the adaptor comprises a first and a second adaptor, wherein the first adaptor is located on the first end of the double stranded polynucleotide of interest and wherein the second adaptor is located on the second end of the double stranded polynucleotide of interest upon formation of the circular nucleic acid molecule.

65. The method of claim 64, wherein the first adaptor is connected to the second adaptor through a linking polynucleotide.

66. The method of claim 65, wherein upon formation of the circular nucleic acid molecule, the first nick is located at an end of the first adaptor that is distal to an end of the first adaptor that is connected to the double stranded polynucleotide of interest and the second nick is located at an end of the second adaptor that is distal to an end of the second adaptor that is connected to the double stranded polynucleotide of interest.

67. The method of claim 66, wherein the adaptors are ligated to a linking polynucleotide before the adaptors are ligated to the double stranded polynucleotide of interest.

68. The method of claim 61, further comprising digesting at least a part of the first strand of the circular nucleic acid molecule and digesting at least a part of the second strand of the circular nucleic acid molecule, wherein the digesting results in at least a portion of the double stranded polynucleotide of interest becoming single stranded, wherein the digesting occurs before the extending of the first and second 3′

ends.

69. The method of claim 68, wherein a 5′

to 3′

exonuclease is used to digest said nicked circular nucleic acid molecule.

70. The method of claim 68, wherein the digestion is halted before the double stranded polynucleotide of interest becomes single stranded in its entirety.

71. The method of claim 68, wherein the entire double stranded polynucleotide of interest is digested to a single stranded polynucleotide of interest.

72. The method of claim 68, wherein a DNA polymerase is used to extend said nicked circular nucleic acid molecule.

73. The method of claim 61, further comprising digesting at least a part of the first strand of the circular nucleic acid molecule and digesting at least a part of the second strand of the circular nucleic acid molecule, wherein the digesting results in at least a portion of the double stranded polynucleotide of interest becoming single stranded, wherein the digesting occurs at the same time as the extending of the first and second 3′

ends.

74. The method of claim 73, wherein the digesting and the extending take place in the same reaction tube.

75. The method of claim 61, wherein the extending of the first 3′

end of the first strand of the circular nucleic acid molecule into a sequence of the double stranded polynucleotide of interest is achieved by nick translation.

76. The method of claim 75, wherein the extending of the second 3′

end of the second strand of the circular nucleic acid molecule into a sequence of the double stranded polynucleotide of interest is achieved by nick translation.

77. The method of claim 76, wherein the first and second nicks are expanded into a first gap and second gap respectively.

78. The method of claim 77, wherein an exonuclease is used to expand the first and second nicks into the first gap and second gap respectively.

79. The method of claim 78, wherein the exonuclease comprises a T7 exonuclease.

80. The method of claim 61, further comprising performing a single strand dependent digest to release the paired DNA tag.

81. A nicked linking polynucleotide comprising:

a first adaptor comprising a first adaptor strand that is hybridized to a second adaptor strand, wherein the first adaptor strand lacks a phosphate group on its 5′

end;

a second adaptor comprising a third adaptor strand that is hybridized to a fourth adaptor strand, wherein the third adaptor strand lacks a phosphate group on its 5′

end; and

a linking polynucleotide comprising a first linking strand hybridized to a second linking strand, wherein the linking polynucleotide comprises a first end and a second end, wherein the first adaptor is attached to the first end of the linking polynucleotide such that a nick is present where the first adaptor strand lacks a phosphate group on its 5′

end, wherein the second linking strand is attached to the second adaptor strand, wherein the second adaptor is attached to the second end of the linking polynucleotide such that a nick is present where the third adaptor strand of the second adaptor lacks a phosphate group on its 5′

end, wherein the second strand of the linking polynucleotide is attached to the fourth adaptor strand.

82. The nicked linking polynucleotide of claim 81, further comprising a phosphate group on a 5′

end of the second adaptor strand.

83. The nicked linking polynucleotide of claim 82, further comprising a phosphate group on a 5′

end of the fourth adaptor strand.

84. The nicked linking polynucleotide of claim 83, wherein there is no phosphate group on the further comprising a phosphate group on a 3′

end of the second adaptor strand.

85. A nicked linking polynucleotide comprising:

a first double stranded adaptor;

a double stranded linking polynucleotide, wherein the first double stranded adaptor is attached to a first end of a double stranded linking polynucleotide; and

a second double stranded adaptor attached to a second end of the double stranded linking polynucleotide, wherein there is a first nick separating a first strand of the first adaptor and a first strand of the double stranded vector and a second nick separating a second strand of the second adaptor and a first strand of the double stranded vector, wherein the first nick and second nick are on different strands of the nicked vector.

86. A method of preparing a paired tag, said method comprising:

providing a nicked linking polynucleotide comprising;

a first adaptor comprising a first adaptor strand that is hybridized to a second adaptor strand, wherein the first adaptor strand lacks a phosphate group on its 5′

end;

a second adaptor comprising a third adaptor strand that is hybridized to a fourth adaptor strand, wherein the third adaptor strand lacks a phosphate group on its 5′

end; and

a linking polynucleotide comprising a first linking strand hybridized to a second linking strand, wherein the linking polynucleotide comprises a first end and a second end, wherein the first adaptor is attached to the first end of the linking polynucleotide such that a nick is present where the first adaptor strand lacks a phosphate group on its 5′

end, wherein the second linking strand is attached to the second adaptor strand, wherein the second adaptor is attached to the second end of the linking polynucleotide such that a nick is present where the third adaptor strand of the second adaptor lacks a phosphate group on its 5′

end, wherein the second strand of the linking polynucleotide is attached to the fourth adaptor strand;

ligating a first end of a double stranded polynucleotide of interest to the first adaptor and ligating a second end of the double stranded polynucleotide of interest to the second adaptor; and

moving the first and second nicks into the double stranded polynucleotide of interest.

87. The method of claim 86, further comprising a single strand dependent digest to linearize the nicked linking polynucleotide.

88. The method of claim 87, further comprising ligating an internal adaptor into the linearized nicked linking polynucleotide.

89. The method of claim 88, further comprising amplifying at least one strand of the double stranded polynucleotide of interest.

90. The method of claim 89, wherein the amplification is a PCR based amplification.

91. A linear paired tag, said linear paired tag comprising:

a first tag sequence, wherein the first tag sequence comprises a first end of a polynucleotide of interest;

a first adaptor covalently bonded to the first tag sequence; and

a second tag sequence covalently bonded to the first adaptor, wherein the second tag sequence comprises a second end of the polynucleotide of interest, and wherein said linear paired tag lacks a type IIs restriction site and lacks a type III restriction site.