Classification of Nucleic Acid Templates

• US 20100221716A1
• Filed: 12/10/2009
• Published: 09/02/2010
• Est. Priority Date: 12/11/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Turner, Stephen, Lyle, John, Korlach, Jonas, Jia, Lei, Sorenson, Jon, Lee, Jessica, Webster, Dale, Flusberg, Benjamin, Hanes, Jeremiah, Travers, Kevin
• Granted Patent: US 9,175,341 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

Methods, compositions, and systems are provided for characterization of modified nucleic acids. In certain preferred embodiments, single molecule sequencing methods are provided for identification of modified nucleotides within nucleic acid sequences. Modifications detectable by the methods provided herein include chemically modified bases, enzymatically modified bases, abasic sites, non-natural bases, secondary structures, and agents bound to a template nucleic acid.

Claims

1. A method for identifying a modification, the method comprising:

a) providing a template nucleic acid comprising the modification;

b) providing an enzyme capable of processing the template nucleic acid;

c) contacting the template nucleic acid with the enzyme;

d) monitoring processing of the template nucleic acid by the enzyme; and

e) detecting a change in the processing, wherein the change is indicative of the modification, thereby identifying the modification.

3. The method of claim 1, wherein the modification is a methylated cytosine or adenine base.

6. The method of claim 1, wherein the template nucleic acid comprises a first polynucleotide region comprising the modification and a second polynucleotide region complementary to the first polynucleotide region, where the first polynucleotide region and the second polynucleotide region are on a single strand of the template nucleic acid.

7. The method of claim 1, wherein the template nucleic acid is subjected to a treatment to alter the modification prior to the contacting.

9. The method of claim 7, wherein the treatment is glycosylase modification to convert the modification to an abasic site, and further wherein the processing results in the synthesis of a nascent nucleic acid strand into which a non-natural nucleotide analog is incorporated opposite the abasic site.

12. The method of claim 9, wherein the non-natural nucleotide analog is a mixture of incorporatable non-natural nucleotide analogs that are non-incorporatable non-natural nucleotide analogs.

14. The method of claim 7, wherein the treatment is DMS modification and the modification is secondary structure of the template nucleic acid or an agent bound to the template nucleic acid.

16. The method of claim 1, wherein the processing is rolling-circle synthesis of a nascent nucleic acid strand.

18. The method of claim 1, wherein the enzyme is a polymerase enzyme.

22. The method of claim 1, wherein the processing results in the synthesis of a nascent nucleic acid strand.

24. The method of claim 22, wherein the monitoring detects incorporation of single nucleotides into the nascent nucleic acid strand to generate a sequence read that is complementary to the template nucleic acid.

27. The method of claim 24, wherein the change comprises incorporation of a non-natural nucleotide analog at a position in the nascent nucleic acid strand that is complementary to a position in the template nucleic acid that comprises the modification.

28. The method of claim 27, wherein the change further comprises a kinetic difference in the processing.

32. The method of claim 24, wherein the single nucleotides are differentially labeled to be distinguishable from one another during the monitoring.

33. The method of claim 32, wherein the single nucleotides comprise a label linked to a phosphate group, wherein the phosphate group is removed during the processing.

35. The method of claim 24, further comprising mapping the modification within the template nucleic acid, the mapping comprising:

f) analyzing a portion of the sequence read that was generated immediately prior to, during, or immediately after the detecting the change in processing to determine a sequence complementary to the template nucleic acid;

g) determining the complement of the sequence complementary to the template nucleic acid in f; and

h) mapping the modification at a position in the template nucleic acid that is proximal to the complement of the sequence complementary to the template nucleic acid in f.

36. The method of claim 1, wherein the change is a kinetic difference in the processing.

37. The method of claim 36, wherein the kinetic difference is detected as an alteration in interpulse duration during the processing.

38. The method of claim 36, wherein the kinetic difference is detected as an alteration in pulse width during the processing.

39. The method of claim 36, wherein the kinetic difference is indicative of what type of modification is present in the template nucleic acid.

40. The method of claim 1, wherein the monitoring occurs in real time during the processing.

41. The method of claim 1, wherein the template nucleic acid and the enzyme form a complex that is immobilized at a reaction site on a substrate.

42. The method of claim 41, wherein a plurality of complexes are immobilized at optically resolvable reaction sites on the substrate, wherein a single complex immobilized at one of the reaction sites is optically resolvable from any other of the complexes immobilized at any other of the reaction sites.

43. The method of claim 42, wherein the optically resolvable reaction sites are nanometer-scale apertures in the substrate.

48. The method of claim 1, wherein the modification is a secondary structure formation and the change is an alteration in one or more kinetic characteristics of the processing, the method further comprising:

f) generating a sequence read for the template nucleic acid before and after the change;

g) identifying a first portion of the sequence read generated before the change that is complementary to a second portion of the sequence read generated after the change; and

h) determining a likelihood that a secondary structure formed by association of the first portion to the second portion was present in the template nucleic acid during the processing based at least upon the nucleotide composition of the first portion and the second portion.

2. (canceled)

4-5. -5. (canceled)

8. (canceled)

10-11. -11. (canceled)

13. (canceled)

15. (canceled)

17. (canceled)

19-21. -21. (canceled)

23. (canceled)

25-26. -26. (canceled)

29-31. -31. (canceled)

34. (canceled)

44-47. -47. (canceled)

49. A method of detecting binding of an agent to a single nucleic acid template, comprising:

f) providing the single nucleic acid template in complex with a polymerase;

g) introducing a reaction mixture to the complex, wherein the reaction mixture comprises the agent; and

h) monitoring synthesis of a polynucleotide by the polymerase, wherein the polynucleotide is complementary to the single nucleic acid template, and wherein a change in the synthesis is indicative of binding of the agent to the single nucleic acid template.

52. The method of claim 49, further comprising determining a consensus binding site of the agent, said determining comprising:

d) performing a plurality of sequencing-by-synthesis reactions on a set of single nucleic acid templates in the presence of the agent to generate a set of binding-affected nascent polynucleotide sequences;

e) performing a plurality of sequencing-by-synthesis reactions on the set of single nucleic acid templates in the absence of the agent to generate a set of full-length nascent polynucleotide sequences;

f) analyzing the binding-affected nascent polynucleotide sequences to determine a location at which the agent bound the single nucleic acid template during the sequencing-by-synthesis reactions in the presence of the agent; and

g) identifying a sequence common to the full-length nascent polynucleotide sequences at the location determined in (iii), thereby identifying the consensus binding site of the agent.

53. The method of claim 52, wherein the binding-affected nascent polynucleotide sequences are truncated nascent polynucleotide sequences.

54. The method of claim 52, wherein the binding-affected nascent polynucleotide sequences are nascent polynucleotide sequences whose synthesis was paused at the location determined in f.

50-51. -51. (canceled)

55. A method for detecting a modification in a single nucleic acid template during a sequencing-by-synthesis reaction, the method comprising:

a) providing the single nucleic acid template in complex with a polymerase;

b) introducing a reaction mixture to the complex, wherein the reaction mixture comprises an agent that specifically binds to the modification; and

c) monitoring synthesis of a polynucleotide by the polymerase, wherein the polynucleotide is complementary to the single nucleic acid template, and wherein a pause or cessation of the synthesis of the polynucleotide is indicative of binding of the agent to the single nucleic acid template, thereby detecting the modification in the single nucleic acid template.

56. The method of claim 55, wherein the modification is an 8-oxoG lesion.

58. The method of claim 55, wherein the modification is a methylated base.

60. The method of claim 55, wherein the modification is a secondary structure formation in the nucleic acid template.

57. (canceled)

59. (canceled)

61-73. -73. (canceled)

74. A composition comprising:

i) a substrate comprising a reaction site that is optically resolvable from any other reaction site on the substrate;

j) a single complex immobilized at the reaction site, wherein the complex comprises a template nucleic acid and a sequencing engine;

k) a mixture of incorporatable nucleotides or nucleotide analogs; and

l) at least one modification in the template nucleic acid, wherein the template at or proximal to the modification is processed differently than the template distal from the modification.

76. The composition of claim 74, wherein the modification is in a strand of the template nucleic acid that is complementary to a nascent strand synthesized by the sequencing engine.

77. The composition of claim 74, wherein the modification is in a strand of the template nucleic acid that is displaced by the sequencing engine.

78. The composition of claim 74, further comprising at least one type of non-incorporatable nucleotide analog.

81. The composition of claim 74, further comprising an agent other than the sequencing engine that binds to and/or chemically or enzymatically alters the modification.

75. (canceled)

79-80. -80. (canceled)

82-100. -100. (canceled)