DNA sequencing by stepwise ligation and cleavage

• US 5,552,278 A
• Filed: 07/25/1994
• Issued: 09/03/1996
• Est. Priority Date: 04/04/1994
• Status: Expired due to Term

• Current Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Original Assignee: Spectragen, Inc.
• Inventors: Brenner, Sydney
• Primary Examiner(s): SHOEMAKER, DEBRA GUNNERSEN
• Assistant Examiner(s): Tran, Paul B.
• Application Number:
  US08/280,441
• Time in Patent Office: 771 Days
• Field of Search: 435/6, 435/18, 435/91.52, 435/91.53, 536/24.3, 935/77, 935/78
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• 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 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: Y02P 20/582 Recycling of unreacted starting or intermediate materials

Abstract

The invention provides a method of nucleic acid sequence analysis based on repeated cycles of ligation to and cleavage of probes at the terminus of a target polynucleotide. At each such cycle one or more terminal nucleotides are identified and one or more nucleotides are removed from the end of the target polynucleotide, such that further cycles of ligation and cleavage can take place. At each cycle the target sequence is shortened by one or more nucleotides until the nucleotide sequence of the target polynucleotide is determined. The method obviates electrophoretic separation of similarly sized DNA fragments and eliminates the difficulties associated with the detection and analysis of spacially overlapping bands of DNA fragments in a gel, or like medium. The invention further obviates the need to generate DNA fragments from long single stranded templates with a DNA polymerase.

Claims

1. A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of:

(a) providing the polynucleotide in double stranded form such that the polynucleotide has a protruding strand at at least one end;

(b) ligating a probe from a mixture of probes to an end of the polynucleotide having a protruding strand to form a ligated complex, each probe having an end with a complementary protruding strand to that of the polynucleotide and each probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site;

(c) identifying one or more nucleotides in the protruding strand of the polynucleotide;

(d) cleaving the ligated complex with said nuclease that recognizes said nuclease recognition site and cuts the ligated complex to give an augmented probe and a new protruding strand on the polynucleotide; and

(e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined.

2. The method of claim 1 wherein the step of providing further includes blocking the recognition sites of said nuclease on said polynucleotide.

3. The method of claim 2 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.

4. The method of claim 3 wherein said step of ligating includes treating said polynucleotide with a ligase.

5. The method of claim 4 wherein said polynucleotide contains a 5'"'"'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'"'"'-phosphoryl group on said end having said complementary protruding strand.

6. The method of claim 5 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.

7. The method of claim 1 further including the step of removing unligated probe from said ligated complex after said step of ligating.

8. The method of claim 1 wherein said step of identifying includes identifying said one or more nucleotides in said protruding strand of said polynucleotide by the identity of said ligated probe.

9. The method of claim 1 wherein said protruding strand of said polynucleotide includes a 3'"'"' recessed strand and wherein step of identifying includes extending the 3'"'"' recessed strand by a nucleic acid polymerase in the presence of at least one chain-terminating nucleoside triphosphate so that at least one chain-terminating nucleoside triphosphate is incorporated into the 3'"'"' recessed strand.

10. The method of claim 9 wherein said chain-terminating nucleoside triphosphate is a labeled dideoxynucleoside triphosphate and wherein said step of identifying includes identifying said one or more nucleotides by the label on the labeled dideoxynucleoside triphosphate incorporated into said 3'"'"' recessed strand of said polynucleotide as a labeled dideoxynucleotide.

11. The method of claim 10 further including the steps of excising said labeled dideoxynucleotide and extending said recessed strand with a nucleic acid polymerase.

12. A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of:

(a) ligating a probe from a mixture of probes to an end of a polynucleotide having a protruding strand to forth a ligated complex, each probe having an end with a complementary protruding strand to that of the polynucleotide and each probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site;

(b) removing unligated probe from the ligated complex;

(c) cleaving the ligated complex with said nuclease, said nuclease recognizing the recognition site and cleaving the ligated complex to give an augmented probe and a new protruding strand on the polynucleotide;

(d) identifying one or more nucleotides in the protruding strand of the polynucleotide; and

(e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined.

13. The method of claim 12 further including the step of providing said polynucleotide with said recognition sites of said nuclease blocked.

14. The method of claim 13 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.

15. The method of claim 14 wherein said step of ligating includes treating said polynucleotide with a ligase.

16. The method of claim 15 wherein said polynucleotide contains a 5'"'"'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'"'"'-phosphoryl group on said end having said complementary protruding strand.

17. The method of claim 16 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.

18. A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of:

(a) ligating a probe from a mixture of probes to an end of a polynucleotide having a protruding strand to form a ligated complex, the probe having an end with a complementary protruding strand to that of the polynucleotide and the probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site;

(b) identifying one or more nucleotides in the protruding strand of the polynucleotide;

(c) cleaving the ligated complex with said nuclease so that the polynucleotide is shortened by one or more nucleotides; and

(d) repeating steps (a) through (c) until the nucleotide sequence of said nuclease polynucleotide is determined.

19. The method of claim 18 further including the step of providing said polynucleotide with said recognition sites of said nuclease blocked.

20. The method of claim 19 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.

21. The method of claim 20 wherein said step of ligating includes treating said polynucleotide with a ligase.

22. The method of claim 21 wherein said polynucleotide contains a 5'"'"'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'"'"'-phosphoryl group on said end having said complementary protruding strand.

23. The method of claim 22 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.

24. The method of claim 23 wherein the identity of a single nucleotide is determined in said step of identifying and said polynucleotide is shortened by a single nucleotide in said step of cleaving.

25. The method of claim 18 further including the step of removing unligated probe from said ligated complex after said step of ligating.

26. The method of claim 18 wherein said step of identifying includes identifying said one or more nucleotides in said protruding strand of said polynucleotide by the identity of said ligated probe.

27. The method of claim 18 wherein said protruding strand of said polynucleotide includes a 3'"'"' recessed strand and wherein step of identifying includes extending the 3'"'"' recessed strand by a nucleic acid polymerase in the presence of at least one chain-terminating nucleoside triphosphate so that at least one chain-terminating nucleoside triphosphate is incorporated into the 3'"'"' recessed strand.

28. The method of claim 27 wherein said chain-terminating nucleoside triphosphate is a labeled dideoxynucleoside triphosphate and wherein said step of identifying includes identifying said one or more nucleotides by the label on the labeled dideoxynucleoside triphosphate incorporated into said 3'"'"' recessed strand of said polynucleotide.

29. A method for determining the identity of a terminal nucleotide of a polynucleotide, the method comprising the steps of:

(a) ligating a probe from a mixture to an end of a polynucleotide having a protruding strand to form a ligated complex, the probe having an end with a complementary protruding strand to that of the polynucleotide and the probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site;

(b) removing unligated probe from the ligated complex; and

(c) identifying the terminal nucleotide of the polynucleotide by the identity of the ligated probe.

30. A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of:

(a) providing the polynucleotide in double stranded form such that the polynucleotide has a protruding strand at at least one end;

(b) ligating a probe to an end of the polynucleotide having a protruding strand to form a ligated complex, the probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site and an end with a complementary protruding strand to that of the polynucleotide;

(c) identifying one or more nucleotides in the protruding strand of the polynucleotide;

(d) cleaving the ligated complex with a nuclease that recognizes the recognition site and cuts the ligated complex to give an augmented probe and a new protruding strand on the polynucleotide; and

(e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined.

31. The method of claim 30 wherein the step of providing further includes blocking the recognition sites of said nuclease on said polynucleotide.

32. The method of claim 31 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.

33. The method of claim 32 wherein said step of ligating includes treating said polynucleotide with a ligase.

34. The method of claim 33 wherein said polynucleotide contains a 5'"'"'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'"'"'-phosphoryl group on said end having said complementary protruding strand.

35. The method of claim 34 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.

36. The method of claim 30 further including the step of removing unligated probe from said ligated complex after said step of ligating.

37. The method of claim 30 wherein said step of identifying includes identifying said one or more nucleotides in said protruding strand of said polynucleotide by the identity of said ligated probe.

38. The method of claim 30 wherein said protruding strand of said polynucleotide includes a 3'"'"' recessed strand and wherein step of identifying includes extending the 3'"'"' recessed strand by a nucleic acid polymerase in the presence of at least one chain-terminating nucleoside triphosphate so that at least one chain-terminating nucleoside triphosphate is incorporated into the 3'"'"' recessed strand.

39. The method of claim 38 wherein said chain-terminating nucleoside triphosphate is a labeled dideoxynucleoside triphosphate and wherein said step of identifying includes identifying said one or more nucleotides by the label on the labeled dideoxynucleoside triphosphate incorporated into said 3'"'"' recessed strand of said polynucleotide as a labeled dideoxynucleotide.

40. The method of claim 39 further including the steps of excising said labeled dideoxynucleotide and extending said recessed strand with a nucleic acid polymerase.

41. A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of:

(a) ligating a probe to an end of a polynucleotide having a protruding strand to form a ligated complex, the probe having an end with a complementary protruding strand to that of the polynucleotide and the probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site;

(b) removing unligated probe from the ligated complex;

(c) cleaving the ligated complex with a nuclease, the nuclease recognizing the recognition site and cleaving the ligated complex to give an augmented probe and a new protruding strand on the polynucleotide;

(d) identifying one or more nucleotides in the protruding strand of the polynucleotide; and

(e) repeating steps (a) through (d) until the nucleotide sequence of the polynucleotide is determined.

42. The method of claim 41 further including the step of providing said polynucleotide with said recognition sites of said nuclease blocked.

43. The method of claim 42 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.

44. The method of claim 43 wherein said step of ligating includes treating said polynucleotide with a ligase.

45. The method of claim 44 wherein said polynucleotide contains a 5'"'"'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'"'"'-phosphoryl group on said end having said complementary protruding strand.

46. The method of claim 45 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.

47. A method for determining the nucleotide sequence of a polynucleotide, the method comprising the steps of:

(a) ligating a probe to an end of a polynucleotide having a protruding strand to form a ligated complex, the probe having an end with a complementary protruding strand to that of the polynucleotide and the probe having a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site;

(b) identifying one or more nucleotides in the protruding strand of the polynucleotide;

(c) cleaving the ligated complex with said nuclease so that the polynucleotide is shortened by one or more nucleotides; and

(d) repeating steps (a) through (c) until the nucleotide sequence of the polynucleotide is determined.

48. The method of claim 47 further including the step of providing said polynucleotide with said recognition sites of said nuclease blocked.

49. The method of claim 48 wherein said nuclease is a type IIs restriction endonuclease and wherein said blocking said recognition sites includes treating said polynucleotide with a methylase.

50. The method of claim 49 wherein said step of ligating includes treating said polynucleotide with a ligase.

51. The method of claim 50 wherein said polynucleotide contains a 5'"'"'-phosphoryl group on said end having said protruding strand and said probe lacks a 5'"'"'-phosphoryl group on said end having said complementary protruding strand.

52. The method of claim 51 wherein said step of ligation includes treating said polynucleotide in succession with a ligase, a kinase, and a ligase.

53. The method of claim 52 wherein the identity of a single nucleotide is determined in said step of identifying and said polynucleotide is shortened by a single nucleotide in said step of cleaving.

54. The method of claim 53 further including the step of removing unligated probe from said ligated complex after said step of ligating.

55. The method of claim 47 wherein said step of indentifying includes identifying said one or more nucleotides in said protruding strand of said polynucleotide by the identity of said ligated probe.

56. The method of claim 47 wherein said protruding strand of said polynucleotide includes a 3'"'"' recessed strand and wherein step of identifying includes extending the 3'"'"' recessed strand by a nucleic acid polymerase in the presence of at least one chain-terminating nucleoside triphosphate so that at least one chain-terminating nucleoside triphosphate is incorporated into the 3'"'"' recessed strand.

57. The method of claim 56 wherein said chain-terminating nucleoside triphosphate is a labeled dideoxynucleoside triphosphate and wherein said step of identifying includes identifying said one or more nucleotides by the label on the labeled dideoxynucleoside triphosphate incorporated into said 3'"'"' recessed strand of said polynucleotide.