Paired end sequencing

• US 20060292611A1
• Filed: 06/06/2006
• Published: 12/28/2006
• Est. Priority Date: 06/06/2005
• Status: Active Grant

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Hutchison, Stephen Kyle, Egholm, Michael, Godwin, Brian C., Berka, Jan, Chen, Zhoutao, Sarkis, Gary James, Simons, Jan Fredrik, Leamon, John Harris
• Granted Patent: US 7,601,499 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/125 Methyl transferase, i.e. methylase
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/155 Cyclic reactions
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The present invention provides for a method of preparing a target nucleic acid fragments to produce a smaller nucleic acid which comprises the two ends of the target nucleic acid. Specifically, the invention provides cloning and DNA manipulation strategies to isolate the two ends of a large target nucleic acid into a single small DNA construct for rapid cloning, sequencing, or amplification.

Claims

1. A method for obtaining a DNA construct comprising two end regions of a target nucleic acid comprising the steps of:

(a) fragmenting a large nucleic acid molecule to produce a target nucleic acid;

(b) ligating a capture element to said target nucleic acid to form a first circular nucleic acid molecule;

(c) digesting said first circular nucleic acid with a restriction endonuclease which cuts said target nucleic acid but which does not cut said capture element to produce a linear nucleic acid which comprises two ends of said target nucleic acid separated by said capture element;

(d) ligating said linear nucleic acid with a separator element to form a second circular nucleic acid;

(e) converting said second circular nucleic acid to a circular single strand nucleic acid;

(f) annealing a first oligonucleotide to said circular single stranded nucleic acid and amplifying said circular single stranded nucleic acid by rolling circle amplification to produce a single stranded rolling circle amplification product;

(g) annealing a second oligonucleotide to said single stranded rolling circle amplification product to form multiple double stranded regions in said single stranded rolling amplification product; and

(h) digesting said single stranded rolling circle amplification product into small fragments with a restriction endonuclease which cleaves said multiple double stranded regions to produce said DNA construct comprising two end regions of a target nucleic acid.

2. The method of claim 1 further comprising the step of size fractionating said target nucleic acid fragment and selecting a preferred size of target nucleic acid fragments after said fragmenting step.

3. The method of claim 1 wherein said first oligonucleotide anneals to said capture element or said separator element.

4. The method of claim 1 wherein said second oligonucleotide anneals to said capture element or said separator element.

5. The method of claim 1 wherein said restriction endonuclease is a type I or type IIS restriction endonuclease.

6. The method of claim 1 wherein said target nucleic acid is at least 50 kb, at least 20 kb, at least 10 kb or at least 5 kb.

7. The method of claim 1 wherein said target nucleic acid is between 50 kb and 3 kb, between 20 and 3 kb, or between 10 kb and 3 kb.

8. The method of claim 1 wherein said capture element or said separator element comprises a marker gene.

9. The method of claim 8 wherein said marker gene is an antibiotic resistance gene.

10. The method of claim 1 wherein said capture element or said separator element comprises a eukaryotic or prokaryotic origin of replication.

11. The method of claim 1 wherein said capture element or said separator element is biotinylated.

12. The method of claim 11 further comprising the step of isolating nucleic acid fragments comprising a capture element after said digesting step.

21. The method of claim 1 wherein said adaptors or said signature tag is biotinylated.

22. The method of claim 21 further comprising the step of isolating nucleic acid fragments comprising a signature tag or adaptors after said digesting step.

13. A method for obtaining a DNA construct comprising two end regions of a target nucleic acid comprising the steps of:

(a) fragmenting a large nucleic acid molecule to produce a target nucleic acid;

(b) ligating an adaptor to each end of said target nucleic acid;

(c) ligating a signature tag to said target nucleic acid to form a circular nucleic acid molecule;

(d) digesting said circular nucleic acid with a restriction endonuclease which cuts said target nucleic acid but which does not cut said adaptor or said signature tag to produce said DNA construct comprising two end region of a target nucleic acid.

14. The method of claim 13 further comprising the step of size fractionating said target nucleic acid fragments and selecting a preferred size of target nucleic acid fragment after said fragmenting step.

15. The method of claim 13 further comprise the step of amplifying said DNA construct after said step (d).

16. The method of claim 15 wherein said amplification is performed by

(e) ligating adaptors to the ends of said DNA construct;

and

(f) amplifying said DNA construct by PCR.

17. The method of claim 13 wherein said restriction endonuclease is a type I or type IIS restriction endonuclease.

18. The method of claim 13 wherein said target nucleic acid is at least 50 kb, at least 20 kb, at least 10 kb or at least 5 kb.

19. The method of claim 13 wherein said target nucleic acid is between 50 kb and 3 kb, between 20 and 3 kb, or between 10 kb and 3 kb.

20. The method of claim 13 wherein said signature tag comprises a marker gene or an origin of replication.

75. The method of either one of claims 17, 46 or 74, wherein the restriction endonuclease is MmeI.

76. The method of claim 75, wherein a carrier DNA containing MmeI restriction sites is added during the MmeI digestion.

77. The method of claim 76, wherein the amount of carrier DNA is added in molar excess of the circular nucleic acid.

78. The method of claim 76, wherein MmeI enzyme and MmeI sites in the carrier DNA are present in stoichiometric amounts.

23. A method for obtaining a DNA construct comprising two end regions of a target nucleic acid comprising the steps of:

(a) fragmenting a large nucleic acid molecule to produce a target nucleic acid;

(b) ligating a first adaptor one end of said target nucleic acid and a second adaptor to a second end of said target nucleic acid to form an adaptor tagged target nucleic acid;

(c) circularizing said adaptor tagged target nucleic acid by ligating said first adaptor to said second adaptor to form a circular nucleic acid molecule comprising a target nucleic acid region and an adaptor region;

(d) fragmenting said circular nucleic acid molecule at the target nucleic acid region to produce said DNA construct comprising two end regions of a target nucleic acid.

24. The method of claim 23 wherein said large nucleic acid or said target nucleic acid is methylated with a methylase before step (b).

25. The method of claim 24 wherein the methylation prevents restriction endonuclease cleavage of the target nucleic acid by one or more restriction endonucleases.

26. The method of claim 23 wherein said adaptors are hairpin adaptors.

27. The method of claim 26 further comprising the following steps after step (b):

(b

     1) treating said adaptor tagged target nucleic acid with an exonuclease to digest any target nucleic acids not ligated to hairpin adaptors at both ends;

(b2) removing said exonuclease from said adaptor tagged target nucleic acid.

28. The method of claim 23 further comprising the following step between step (b) and step (c):

(b3) digesting said adaptor tagged target nucleic acid with a restriction endonuclease which cleaves said first and second adaptors and which does not cleave said target nucleic acid to generate an adaptor tagged target nucleic acid with cleaved adaptors at both ends;

29. The method of claim 23 further comprising a step of removing uncircularized target nucleic acid after step (c).

30. The method of claim 29 wherein removing the uncircularized target nucleic acid comprises contacting said target nucleic acid with exonuclease.

31. The method of claim 23 wherein step (d) is performed by mechanical shearing.

32. The method of claim 23 wherein said adaptor is biotinylated.

33. The method of claim 32 further comprising the step of purifying said adaptor tagged target nucleic acid by affinity purification with an avidin or streptavidin coated solid support after step (b).

34. The method of claim 32 further comprising the step of purifying said DNA construct by affinity purification with an avidin or streptavidin coated solid support after step (d).

35. The method of claim 23 wherein said target nucleic acid is at least 50 kb, at least 20 kb, at least 10 kb or at least 5 kb.

36. The method of claim 23 wherein said target nucleic acid is between 50 kb and 3 kb, between 20 and 3 kb, or between 10 kb and 3 kb.

37. The method of claim 23 wherein said target nucleic acid is between at least 500 bp to 1 kb, between 1 kb and 3 kb or between 500 bp and 3 kb.

38. The method of claim 23 wherein said DNA construct-comprising two end regions of a target nucleic acid is less than 10 kb in size.

39. The method of claim 23 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 20 kb in size.

40. The method of claim 23 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 40 kb in size.

41. The method of claim 23 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 5 kb in size.

42. The method of claim 23 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 3 kb in size.

43. The method of claim 23 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 1 kb in size.

44. The method of claim 23 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 500 bp in size.

45. The method of claim 23 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 300 bp in size.

74. The method of claim 23 wherein in step (d), said circular nucleic acid molecule is fragmented by digestion with a restriction endonuclease.

79. The method of claim 26 further comprising the following step between step (b) and step (c):

(b4) digesting said adaptor tagged target nucleic acid with an endonuclease which cleaves said first and second hairpin adaptors and which does not cleave said target nucleic acid to generate an adaptor tagged target nucleic acid with cleaved adaptors at both ends.

80. The method of claim 79 wherein the hairpin adaptors have at least one deoxyinosine in each strand of their double-stranded regions, and wherein the endonuclease is Endonuclease V.

46. A method for obtaining a DNA construct comprising two end regions of a target nucleic acid comprising the steps of:

(a) fragmenting a large nucleic acid molecule to produce a target nucleic acid;

(b) ligating a capture element to said target nucleic acid to form a circular nucleic acid molecule;

(c) digesting said circular nucleic acid with a restriction endonuclease which cuts said target nucleic acid but which does not cut said capture element to produce said DNA construct comprising two end regions of a target nucleic acid separated by said capture element.

47. The method of claim 46 wherein said capture element is a nucleic acid comprising one member of a binding pair.

48. The method of claim 47 wherein said binding pair is selected from the group consisting of FLAG/anti FLAG antibody, biotin/avidin, and biotin/streptavidin.

49. The method of claim 47 wherein said capture element is biotinylated.

50. The method of claim 47 further comprising the step of enriching for said DNA construct by affinity purification using a solid support comprising a second member of said binding pair.

51. The method of claim 46 wherein said large nucleic acid or said target nucleic acid is methylated with a methylase before step (b).

52. The method of claim 51 wherein the methylation prevents restriction endonuclease cleavage of the target nucleic acid by one or more restriction endonucleases.

53. The method of claim 46 further comprising a step of removing uncircularized target nucleic acid after step (b).

54. The method of claim 53 wherein removing the uncircularized target nucleic acid comprises contacting said target nucleic acid with an exonuclease.

55. The method of claim 46 wherein said target nucleic acid is at least 50 kb, at least 20 kb, at least 10 kb or at least 5 kb.

56. The method of claim 46 wherein said target nucleic acid is between 50 kb and 3 kb, between 20 and 3 kb, or between 10 kb and 3 kb.

57. The method of claim 46 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 5 kb in size.

58. The method of claim 46 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 3 kb in size.

59. The method of claim 46 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 1 kb in size.

60. The method of claim 46 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 500 bp in size.

61. The method of claim 46 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 300 bp in size.

62. The method of claim 46 wherein said target nucleic acid is between at least 500 bp to 1 kb, between 1 kb and 3 kb or between 500 bp and 3 kb.

63. The method of claim 46 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 10 kb in size.

64. The method of claim 46 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 20 kb in size.

65. The method of claim 46 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 40 kb in size.

66. A method of circularizing a linear polynucleotide molecule, the method comprising:

a) providing the linear polynucleotide molecule in an aqueous solution;

b) emulsifying the aqueous solution in oil; and

c) covalently joining the ends of the linear polynucleotide molecule, thereby circularizing the linear polynucleotide molecule.

67. The method of claim 66, wherein the linear polynucleotide molecule is selected from the group consisting of single stranded DNA, double stranded DNA, single stranded RNA and double stranded RNA.

68. The method according to claim 66, wherein the two ends of the linear polynucleotide molecule have compatible ends suitable for ligation.

69. The method according to claim 66, wherein the ends of the linear polynucleotide molecule are joined by ligation.

70. The method according to claim 66, wherein the oil is mixed with one or more surfactants.

71. A method of circularizing linear polynucleotide molecules, the method comprising:

a) providing a population of linear polynucleotide molecules in an aqueous solution;

b) emulsifying the aqueous solution in oil to produce a plurality of aqueous microreactors; and

c) covalently joining the ends of the linear polynucleotide molecules in at least one microreactor,

thereby circularizing the linear polynucleotide molecules.

72. The method according to claim 71, wherein a plurality of microreactors contains exactly one polynucleotide molecule.

73. The method according to claim 71, wherein the plurality of microreactors comprise a water-in-oil emulsion.