Paired end sequencing

• US 7,601,499 B2
• Filed: 06/06/2006
• Issued: 10/13/2009
• Est. Priority Date: 06/06/2005
• Status: Expired due to Fees

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Berka, Jan, Sarkis, Gary James, Simons, Jan Fredrik, Leamon, John Harris, Hutchison, Stephen Kyle, Egholm, Michael, Godwin, Brian C., Chen, Zhoutao
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US11/448,462
• Publication Number:
  US 20060292611A1
• Time in Patent Office: 1,225 Days
• Field of Search: None
• 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 hairpin adaptor to each end of said target nucleic acids from step (a), digesting with exonuclease to remove nucleic acids with exposed 5′

or 3′

ends and cleaving said hairpin adaptors to create sticky ends for circularization;

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

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

(e) ligating a PCR primer to each end of said DNA construct; and

(f) amplifying said DNA construct by PCR.

2. The method of claim 1 wherein said restriction endonucleases is a type I or type IIS restriction endonucleases.

3. 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.

4. 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.

5. The method of claim 1 wherein said signature tag comprises a marker gene or an origin of replication.

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

7. The method of claim 6 further comprising the step of isolating nucleic acid fragments comprising a signature tag or hairpin adaptors after said digesting step.

44. The method of any one of claims 2, 27 or 43, wherein the restriction endonucleases is MmeI.

45. The method of claim 44, wherein a carrier DNA containing MmeI restriction sites is added during the MmeI digestion.

46. The method of claim 45, wherein the amount of carrier DNA is added in molar excess of the circular nucleic acid.

47. The method of claim 45, wherein MmeI enzyme and MmeI sites in the carrier DNA are present in stoichiometric amounts.

8. 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 hairpin adaptor to one end of said target nucleic acid and a second hairpin adaptor to a second end of said target nucleic acid to form an adaptor tagged target nucleic acid, wherein the first and second hairpin adaptors comprise a hairpin element and at least one hairpin adaptor is biotinylated;

(c) cleaving the hairpin element from the first and second hairpin adaptors of the adaptor tagged nucleic acid producing a cleaved end on the first and second hairpin adaptor;

(d) circularizing said adaptor tagged target nucleic acid by ligating the cleaved end of said first hairpin adaptor to the cleaved end of said second hairpin adaptor to form a circular nucleic acid molecule comprising a target nucleic acid region and an adaptor region;

(e) fragmenting said circular nucleic acid molecule to produce at least one said DNA construct comprising two end regions of a target nucleic acid with a ligated adaptor region therebetween; and

(f) enriching for said DNA construct by affinity purification with an avidin or streptavidin coated solid support.

9. The method of claim 8 wherein said large nucleic acid or said target nucleic acid is methylated with a methylase before step (b).

10. The method of claim 9 wherein the methylation prevents restriction endonuclease cleavage of the target nucleic acid by one or more restriction endonucleases.

11. The method of claim 8 further comprising the following steps after step (b):

(b1) 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.

12. The method of claim 8 wherein cleaving the hairpin elements comprises digesting said adaptor tagged target nucleic acid with a restriction endonucleases 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 the cleaved ends.

13. The method of claim 8 further comprising a step of removing uncircularized target nucleic acid after step (d).

14. The method of claim 13 wherein removing the uncircularized target nucleic acid comprises contacting said target nucleic acid with exonuclease.

15. The method of claim 8 wherein step (e) is performed by mechanical shearing.

16. The method of claim 8 wherein said target nucleic acid is at least 50 kb, at least 20 kb, at least 10 kb or at least 5 kb.

17. The method of claim 8 wherein said target nucleic acid is between 50 kb and 3 kb, between 20 and 3 kb, or between 10 kb and 3 kb.

18. The method of claim 8 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.

19. The method of claim 8 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 10 kb in size.

20. The method of claim 8 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 20 kb in size.

21. The method of claim 8 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 40 kb in size.

22. The method of claim 8 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 5 kb in size.

23. The method of claim 8 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 3 kb in size.

24. The method of claim 8 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 1 kb in size.

25. The method of claim 8 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 500 bp in size.

26. The method of claim 8 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 300 bp in size.

43. The method of claim 8 wherein in step (de), said circular nucleic acid molecule is fragmented by digestion with a restriction endonucleases.

48. The method of claim 12 wherein the hairpin adaptors have at least one deoxyinosine in each strand of their double-stranded regions, and wherein the endonucleases is Endonuclease V.

27. 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 hairpin adaptor to each end of said target nucleic acids from step (a), digesting with exonuclease to remove nucleic acids with exposed 5′

or 3′

ends and cleaving said hairpin adaptors to create sticky ends for circularization;

(c) ligating a capture element to said target nucleic acid to form a circular nucleic acid molecule, wherein said capture element is a nucleic acid comprising one member of a binding pair;

(d) 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; and

(e) enriching for said DNA construct by affinity purification using a solid support comprising a second member of said binding pair.

28. The method of claim 27 wherein said binding pair is selected from the group consisting of FLAG/anti FLAG antibody, biotin/avidin, and biotin/streptavidin.

29. The method of claim 27 wherein said capture element is biotinylated.

30. The method of claim 27 further comprising a step of removing uncircularized target nucleic acid after step (c).

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

32. The method of claim 27 wherein said target nucleic acid is at least 50 kb, at least 20 kb, at least 10 kb or at least 5 kb.

33. The method of claim 27 wherein said target nucleic acid is between 50 kb and 3 kb, between 20 and 3 kb, or between 10 kb and 3 kb.

34. The method of claim 27 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 5 kb in size.

35. The method of claim 27 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 3 kb in size.

36. The method of claim 27 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 1 kb in size.

37. The method of claim 27 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 500 bp in size.

38. The method of claim 27 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 300 bp in size.

39. The method of claim 27 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.

40. The method of claim 27 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 10 kb in size.

41. The method of claim 27 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 20 kb in size.

42. The method of claim 27 wherein said DNA construct comprising two end regions of a target nucleic acid is less than 40 kb in size.

49. 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, wherein said large nucleic acid or said target nucleic acid is methylated with a methylase;

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

(b1) treating said adaptor tagged target nucleic acid with an exonuclease to digest any target nucleic acids not ligated to the hairpin adaptors at both ends;

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

(b3) digesting said adaptor tagged target nucleic acid with a restriction endonuclease which cleaves the hairpins from 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;

(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; and

(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.

50. The method of claim 49, wherein at least one of said first or second adaptor is biotinylated.

51. The method of claim 49, 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 (d).

52. The method of claim 49, wherein at least one of said first or second adaptor additionally comprises a Type IIs restriction site and the fragmenting in step (d) is accomplished with a Type IIs restriction enzyme.

53. The method of claim 49, wherein the fragmenting in step (d) is accomplished with mechanical shearing.