DNA amplification and sequencing using DNA molecules generated by random fragmentation

• US 20030143599A1
• Filed: 11/13/2002
• Published: 07/31/2003
• Est. Priority Date: 11/13/2001
• Status: Active Grant

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Rubicon Genomics, Inc
• Inventors: Makarov, Vladimir L., Kamberov, Emmanuel, Bruening, Eric, Sleptsova, Irina
• Granted Patent: US 7,655,791 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2523/107 Chemical cleaving agents
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

The present invention is directed to methods to prepare a DNA molecule or a plurality of DNA molecules by random fragmentation. In some embodiments, the present invention regards preparing a template for DNA sequencing by random fragmentation. In specific embodiments, the random fragmentation comprises chemical fragmentation, mechanical fragmentation, or enzymatic fragmentation. In further specific embodiments, a universal sequence is attached to the 3′ end of the DNA fragments, such as by ligation of an adaptor sequence or by homopolymeric tailing with terminal deoxynucleotidyltransferase. In other embodiments, a library is prepared with methods of the present invention.

Claims

1. A method of preparing a DNA molecule, comprising:

obtaining a DNA molecule;

randomly fragmenting the DNA molecule to produce DNA fragments;

attaching a primer having substantially known sequence to at least one end of a plurality of the DNA fragments to produce primer-linked fragments; and

amplifying a plurality of the primer-linked fragments.

2. The method of claim 1, further comprising concomitantly sequencing the plurality of primer-linked fragments.

3. The method of claim 1, wherein said randomly fragmenting of the DNA molecule is by mechanical fragmentation.

4. The method of claim 3, wherein said mechanical fragmentation of the DNA is by hydrodynamic shearing, sonication, or nebulization.

5. The method of claim 1, wherein said randomly fragmenting of the DNA molecule is by chemical fragmentation.

6. The method of claim 5, wherein said chemical fragmentation is by acid catalytic hydrolysis, alkaline catalytic hydrolysis, hydrolysis by metal ions, hydroxyl radicals, irradiation, or heating.

7. The method of claim 5, wherein said chemical fragmentation is by heating.

8. The method of claim 5, wherein said heating is to a temperature of between about 40°

C. and 120°

C.

9. The method of claim 5, wherein said heating is to a temperature of between about 80°

C. and 100°

C.

10. The method of claim 5, wherein said heating is to a temperature of between about 90°

C. and 100°

C.

11. The method of claim 5, wherein said heating is to a temperature of between about 92°

C. and 98°

C.

12. The method of claim 5, said heating is to a temperature of between about 93°

C. and 97°

C.

13. The method of claim 5, wherein said heating is to a temperature of between about 94°

C. and 96°

C.

14. The method of claim 5, wherein said heating is to a temperature of about 95°

C.

15. The method of claim 5, wherein said heating of the DNA molecule is in a solution having from 0 to about 100 mM concentration of a salt.

16. The method of claim 5, wherein said heating is in a solution having from about 0 to about 10 mM concentration of salt.

17. The method of claim 5, wherein said heating is in a solution having from about 0.1 to about 1 mM concentration of salt.

18. The method of claim 5, wherein said heating is in a solution having from about 0.1 to about 0.5 mM concentration of salt.

19. The method of claim 5, wherein said heating is in a solution of 10 mM Tris, pH 8.0;

1 mM EDTA.

20. The method of claim 5, wherein said heating is in a solution of water.

21. The method of claim 1, wherein said randomly fragmenting of the DNA molecule is by enzymatic fragmentation.

22. The method of claim 21, wherein said enzymatic fragmentation comprises digestion with DNAse I.

23. The method of claim 22, wherein said DNAse I digestion is in the presence of Mg²⁺ ions.

24. The method of claim 23, wherein the concentration of said Mg²⁺ is about 1 mM to about 10 mM.

25. The method of claim 22, wherein said DNAse I digestion is in the presence of Mn²⁺ ions.

26. The method of claim 25, wherein the concentration of said Mn²⁺ is about 1 mM to about 10 mM.

27. The method of claim 1, wherein said primer is attached to at least one 3′

end of at least one DNA fragment.

28. The method of claim 27, wherein said attachment of a primer having substantially known sequence to at least one 3′

end of at least one DNA fragment comprises generation of a homopolymer extension of said DNA fragment.

29. The method of claim 28, wherein said homopolymeric extension is generated by terminal deoxynucleotidyltransferase.

30. The method of claim 29, wherein said homopolymeric extension comprises a polyG tract.

31. The method of claim 27, wherein said attachment of a substantially known sequence to at least one 3′

end of at least one DNA fragment comprises ligation of an adaptor molecule to at least one end of the DNA fragment.

32. The method of claim 31, wherein said adaptor comprises at least one blunt end.

33. The method of claim 32, wherein said adaptor comprises a single stranded region.

34. The method of claim 1, wherein said method further comprises generation of at least one blunt end of said DNA fragments.

35. The method of claim 34, wherein said blunt end is generated by T4 DNA polymerase, Klenow, or a combination thereof.

55. The method of claim 5, wherein said heating is in a solution of water.

85. The method of claim 1, wherein the obtained DNA molecule comprises genomic DNA, BAC DNA, or plasmid DNA.

36. A method of preparing a library of DNA molecules, comprising:

obtaining a plurality of DNA molecules;

randomly fragmenting at least one of the DNA molecules to produce DNA fragments;

attaching a primer having a substantially known sequence to at least one end of a plurality of the DNA fragments to produce primer-linked fragments; and

amplifying a plurality of the primer-linked fragments.

37. The method of claim 36, further comprising concomitantly sequencing the plurality of primer-linked fragments.

38. The method of claim 36, wherein said randomly fragmenting of the DNA molecule is by mechanical fragmentation.

39. The method of claim 38, wherein said mechanical fragmentation of the DNA is by hydrodynamic shearing, sonication, or nebulization.

40. The method of claim 36, wherein said randomly fragmenting of the DNA molecule is by chemical fragmentation.

41. The method of claim 40, wherein said chemical fragmentation is by acid catalytic hydrolysis, alkaline catalytic hydrolysis, hydrolysis by metal ions or complexes, hydroxyl radicals, irradiation, or heating.

42. The method of claim 40, wherein said chemical fragmentation is by heating.

43. The method of claim 42, wherein said heating is to a temperature of between about 40°

C. and 120°

C.

44. The method of claim 42, wherein said heating is to a temperature of between about 80°

C. and 100°

C.

45. The method of claim 42, wherein said heating is to a temperature of between about 90°

C. and 100°

C.

46. The method of claim 42, wherein said heating is to a temperature of between about 92°

C. and 98°

C.

47. The method of claim 42, said heating is to a temperature of between about 93°

C. and 97°

C.

48. The method of claim 42, wherein said heating is to a temperature of between about 94°

C. and 96°

C.

49. The method of claim 42, wherein said heating is to a temperature of about 95°

C.

50. The method of claim 42, wherein said heating of the DNA molecule is in a solution having from 0 to about 100 mM concentration of a salt.

51. The method of claim 42, wherein said heating is in a solution having from about 0 to about 10 mM concentration of salt.

52. The method of claim 42, wherein said heating is in a solution having from about 0.1 to about 1 mM concentration of salt.

53. The method of claim 42, wherein said heating is in a solution having from about 0.1 to about 0.5 mM concentration of salt.

54. The method of claim 42, wherein said heating is in a solution of 10 mM Tris, pH 8.0;

1 mM EDTA.

56. The method of claim 36, wherein said randomly fragmenting of the DNA molecule is by enzymatic fragmentation.

57. The method of claim 56, wherein said enzymatic fragmentation comprises digestion with DNAse I.

58. The method of claim 57, wherein said DNAse I digestion is in the presence of Mg²⁺ ions.

59. The method of claim 57, wherein said DNAse I digestion is in the presence of Mn²⁺ ions.

60. The method of claim 59, wherein said primer is attached to at least one 3′

end of at least one DNA fragment.

61. The method of claim 57, wherein said attachment of the primer having substantially known sequence to at least one 3′

end of at least one DNA fragment comprises generation of a homopolymer extension of said DNA fragment.

62. The method of claim 61, wherein said homopolymeric extension is generated by terminal deoxynucleotidyltransferase.

63. The method of claim 62, wherein said homopolymeric extension comprises a polyG tract.

64. The method of claim 36, wherein said attachment of a primer having substantially known sequence to at least one 3′

end of at least one DNA fragment comprises ligation of an adaptor molecule to at least one end of the DNA fragment.

65. The method of claim 64, wherein said adaptor comprises at least one blunt end.

66. The method of claim 64, wherein said adaptor comprises a single stranded region.

67. The method of claim 66, wherein said method further comprises generation of at least one blunt end of said DNA fragments.

68. The method of claim 67, wherein said blunt end is generated by T4 DNA polymerase, Klenow, or a combination thereof.

69. A library generated by the method of claim 36.

70. A method of generating a library of DNA templates, comprising:

obtaining a plurality of DNA molecules;

randomly fragmenting the plurality of DNA molecules to produce DNA fragments;

attaching a first primer having substantially known sequence to at least one end of a plurality of the DNA fragments to produce primer-linked fragments; and

amplifying a plurality of the primer-linked fragments, wherein the amplification utilizes;

a second primer complementary to a known sequence in the DNA fragments; and

a third primer complementary to the first primer.

71. The method of claim 70, further comprising the step of sequencing concomitantly said plurality of DNA fragments using a fourth primer complementary to said known sequence in the DNA fragments.

72. The method of claim 71, wherein said fourth primer is said second primer.

73. A library generated by the method of claim 70.

74. A method of sequencing a plurality of DNA fragments concomitantly, comprising:

obtaining a plurality of DNA molecules;

randomly fragmenting the DNA molecules to generate a plurality of DNA fragments having overlapping sequences;

attaching a first primer having a substantially known sequence to at least one end of the plurality of the DNA fragments to produce primer-linked fragments; and

amplifying a plurality of the primer-linked fragments, wherein the amplification utilizes;

a second primer complementary to a known sequence in the DNA fragments; and

a third primer complementary to the first primer; and

sequencing said plurality of DNA fragments using a fourth primer complementary to said known sequence in the DNA fragments.

75. The method of claim 74, wherein said fourth primer is said second primer.

76. A method of sequencing a consecutive overlapping series of nucleic acid sequences, comprising the steps of:

obtaining a plurality of DNA molecules having overlapping sequences;

concomitantly sequencing a first region in said plurality of DNA molecules using a primer complementary to a known sequence in said plurality of DNA molecules; and

concomitantly sequencing a second region in said plurality of DNA molecules using a primer complementary to sequence determined from the sequencing of the first region,

wherein the next consecutive sequencing of a region in the overlapping series of nucleic acid sequences is produced by initiating sequencing from the sequence obtained in a preceding overlapping sequencing product.

77. The method of claim 76, wherein said obtaining step is further defined as randomly fragmenting at least one parent DNA molecule to generate a plurality of DNA fragments having overlapping sequences;

attaching a first primer having a substantially known sequence to at least one end of the plurality of the DNA fragments to produce primer-linked fragments; and

amplifying a plurality of the primer-linked fragments, wherein the amplification utilizes;

a second primer complementary to a known sequence in the DNA fragments; and

a third primer complementary to the first primer.

78. A method of sequencing a plurality of DNA molecules, comprising:

obtaining said plurality of DNA molecules by randomly fragmenting a parent DNA molecule;

sequencing concomitantly said plurality of DNA molecules with a primer complementary to a known sequence in said plurality of molecules.

79. The method of claim 78, wherein said method further comprises amplification of the plurality of DNA molecules.

80. The method of claim 79, wherein said amplification is further defined as:

attaching a first primer having a substantially known sequence to at least one end of the plurality of the DNA fragments to produce primer-linked fragments; and

amplifying a plurality of the primer-linked fragments, wherein the amplification utilizes;

a second primer complementary to a known sequence in the DNA fragments; and

a third primer complementary to the first primer.

81. A method of preparing a DNA molecule having sequences that generate secondary structure in said molecule, comprising:

obtaining the DNA molecule having said sequences;

randomly fragmenting the DNA molecule to produce a plurality of DNA fragments, wherein the plurality of DNA fragments comprises DNA fragments having part or all of the sequences which generate the secondary structure;

attaching a primer having substantially known sequence to at least one end of a plurality of the DNA fragments to produce primer-linked fragments; and

amplifying a plurality of the primer-linked fragments.

82. The method of claim 81, further comprising concomitantly sequencing the plurality of primer-linked fragments.

83. The method of claim 81, wherein said plurality of DNA fragments further comprises DNA fragments having none of the sequences which generate the secondary structure.

84. The method of claim 81, wherein said secondary structure is a hairpin, a G quartet, or a triple helix.

86. A method of conditioning a 3′

end of a DNA molecule, comprising exposing said 3′

end to terminal deoxynucleotidyltransferase.

87. The method of claim 86, wherein said terminal deoxynucleotidyltransferase is further defined as comprising 3′

exonuclease activity.

88. The method of claim 86, wherein said exposing step further comprises providing a guanine ribonucleotide, guanine deoxyribonucleotide, or both.

89. A method of providing 3′

exonuclease activity to the end of a DNA molecule comprising the step of introducing terminal deoxynucleotidyltransferase to the end of said molecule.

90. The method of claim 89, wherein said introducing step further comprises providing a guanine ribonucleotide, guanine deoxyribonucleotide, or both.

91. A method of preparing a probe, comprising:

obtaining at least one DNA molecule;

randomly fragmenting the DNA molecule to produce DNA fragments;

attaching a labeled primer having substantially known sequence to at least one end of a plurality of the DNA fragments to produce labeled primer-linked fragments; and

amplifying a plurality of the primer-linked fragments.

92. The method of claim 91, wherein said attaching step of a labeled primer comprises generation of a homopolymer extension of said DNA fragment, wherein said extension comprises the label.

93. The method of claim 92, wherein said homopolymeric extension is generated by terminal deoxynucleotidyltransferase.

94. The method of claim 91, wherein said attaching step of a labeled primer comprises ligation of an adaptor molecule to at least one end of the DNA fragment, wherein the adaptor molecule comprises the label.

95. The method of claim 91, wherein the label comprises a radionuclide, an affinity tag, a hapten, an enzyme, a chromophore, or a fluorophore.

96. A labeled probe generated from the method of claim 91.

97. A kit comprising a probe generated from the method of claim 91.

98. A method of repairing a 3′

end of at least one single stranded DNA molecule, comprising providing to said 3′

end a terminal deoxynucleotidyltransferase.

99. The method of claim 98, wherein said providing step further comprises providing a guanine ribonucleotide, guanine deoxyribonucleotide, or both.

100. A kit for repairing a 3′

end of at least one single stranded DNA molecule, wherein said kit comprises a terminal deoxynucleotidyltransferase.

101. The kit of claim 100, wherein said kit comprises a guanine ribonucleotide, guanine deoxyribonucleotide, or both.

102. A method of detecting a damaged DNA molecule, comprising the step of providing to said damaged DNA molecule terminal deoxynucleotidyltransferase and a labeled guanine ribonucleotide, labeled guanine deoxyribonucleotide, or both.

103. The method of claim 102, wherein the damaged DNA molecule comprises a nick, a double stranded break, or both.

104. The method of claim 102, wherein the providing step is further defined as providing repair to said damaged DNA molecule.

105. The method of claim 102, wherein said label comprises a radionuclide, an affinity tag, a hapten, an enzyme, a chromophore, or a fluorophore.

106. The method of claim 102, wherein said damaged DNA is outside a cell.

107. The method of claim 106, wherein said damaged DNA is the result of radiation, ultraviolet light, oxygen, a radical, a metal ion, a nuclease, or mechanical force.

108. The method of claim 102, wherein said damaged DNA is in a cell.

109. The method of claim 108, wherein said cell is an apoptotic cell.

110. The method of claim 108, wherein said damaged DNA is the result of radiation, heat, ultraviolet light, oxygen, radicals, nitric oxide, catecholamine, or a nuclease.