CLASSIFICATION OF NUCLEIC ACID TEMPLATES

• US 20110183320A1
• Filed: 11/12/2010
• Published: 07/28/2011
• 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: Tomaney, Austin B., Puglisi, Joseph, Heiner, Cheryl, Lee, Jessica, Marks, Patrick, Kislyuk, Andrey, Vilfan, Igor, Webster, Dale, Flusberg, Benjamin, Clark, Tyson A., Travers, Kevin, Wegener, Jeffrey, Turner, Stephen, Lyle, John, Korlach, Jonas, Jia, Lei, Sorenson, Jon, Hanes, Jeremiah
• Granted Patent: US 9,175,338 B2
• Time in Patent Office: Days
• US Class Current: 435/6.1
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/689 for bacteria
• 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 2600/154 Methylation markers

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) introducing a further modification into the template nucleic acid;

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

d) contacting the template nucleic acid with the enzyme;

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

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

2. The method of claim 1, wherein the modification is selected from the group consisting of a methylated base, a hydroxymethylated base;

HOMedU, β

-D-glucosyl-HOMedU, cytosine-5-methylenesulfonate, a pseudouridine base, an 7,8-dihydro-8-oxoguanine base, a 2′

-O-methyl derivative base, a nick, an apurinic site, an apyrimidic site, a pyrimidine dimer, a cis-platen crosslinking, oxidation damage, hydrolysis damage, a bulky base adduct, a thymine dimer, a photochemistry reaction product, an interstrand crosslinking product, a mismatched base, a secondary structure, and a bound agent.

3. The method of claim 1, wherein the template nucleic acid comprises a single-stranded portion and a double-stranded portion.

5. 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.

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

7. The method of claim 6, wherein the treatment comprises exposure to a modifying agent selected from the group consisting of a glycosylase, bisulfite, DMS, a cytosine methyltransferase, a hydroxylase, a restriction enzyme, a glucosyltrasferase, NMIA, CDI, and a cytidine deaminase.

9. The method of claim 6, wherein the treatment comprises addition of a sugar moiety to a nucleobase comprising the modification, wherein the change in the processing is greater than in the absence of the sugar moiety.

10. The method of claim 9, wherein the nucleobase is a hydroxymethylcytosine nucleobase, which is converted to β

-glucosyl-5-hydroxymethylcytosine by the addition of the sugar moiety.

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

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

25. The method of claim 1, wherein the processing is a single-molecule sequencing reaction.

26. The method of claim 24, 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 26, wherein the change occurs at the modification and/or at one or more positions upstream or downstream of the modification.

32. The method of claim 26, further comprising mapping the modification within the template nucleic acid, the mapping comprising:

g) 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;

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

i) 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.

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

43. The method of claim 1, wherein the template nucleic acid is a plurality of template nucleic acids that are optically resolvable from one another during the monitoring.

4. (canceled)

8. (canceled)

11-19. -19. (canceled)

21-23. -23. (canceled)

28-31. -31. (canceled)

33-37. -37. (canceled)

39-42. -42. (canceled)

44. (canceled)

45. A method of mapping a binding site of a binding agent within a single nucleic acid template, comprising:

g) exposing the single nucleic acid template to the binding agent;

h) subjecting the single nucleic acid template to template-directed synthesis of a nascent strand complementary to the single nucleic acid template; and

i) monitoring the synthesis of the nascent strand, wherein a locus within the single nucleic acid template at which a change in the synthesis of the nascent stand occurs is identified as being within or adjacent to a binding site of the binding agent within the single nucleic acid template, thereby mapping the binding site.

46. The method of claim 45, wherein the binding agent is selected from the group consisting of a transcription factor, a polymerase, a reverse transcriptase, a histone, a restriction enzyme, an antibody, a nucleic acid binding protein, a nucleic acid binding agent, and a nucleic acid damage binding agent.

48. The method of claim 45, further comprising crosslinking the binding agent to the single nucleic acid template prior to the template-directed synthesis.

49. The method of claim 48, wherein subsequent to said crosslinking and prior to the template-directed synthesis, the binding agent is removed from the single nucleic acid template.

55. The method of claim 45, wherein the binding agent is linked to a modifying agent that introduces a modification into the single nucleic acid template proximal to the binding site, and further wherein the modification causes the change in the synthesis.

56. The method of claim 55, wherein the modifying agent comprises a reactant selected from the group consisting of a methyltransferase, a glycosylase, a glucosytransferase, a hydroxylase, and a nucleic acid damaging agent.

47. (canceled)

50-54. -54. (canceled)

57-64. -64. (canceled)

65. A method of mapping a modification using a single nucleic acid sample, the method comprising:

a) providing a single nucleic acid sample comprising multiple nucleic acid molecules comprising a modification at a particular locus;

b) removing an aliquot of the single nucleic sample, wherein the aliquot comprises some of the nucleic acid molecules comprising the modification;

c) subjecting the aliquot to an amplification reaction, wherein the resulting amplicons do not comprise the modification;

d) determining the nucleotide sequence of the particular locus in the resulting amplicons;

e) determining the nucleotide sequence of the particular locus in a portion of the single nucleic acid sample that was not in the aliquot; and

f) analyzing the nucleotide sequences determined in d and e to map the modification in the single nucleic acid sample.

67. The method of claim 65, wherein prior to step b an enrichment procedure is performed, the enrichment procedure comprising:

g) exposing the single nucleic acid sample to a binding agent that specifically associates with the modification under conditions that promote such an association;

h) immobilizing the binding agent, thereby immobilizing nucleic acids comprising the modification bound to the binding agent from nucleic acids that do not comprise the modification bound to the binding agent;

i) removing the nucleic acids that do not comprise the modification bound to the binding agent while retaining the nucleic acids comprising the modification bound to the binding agent, thereby isolating the nucleic acids comprising the modification bound to the binding agent; and

j) releasing the nucleic acids comprising the modification such that the single nucleic acid sample is now enriched for the nucleic acids comprising the modification.

68. The method of claim 65, further comprising:

g) prior to step d, adding a first barcode to the resulting amplicons

h) prior to step e, adding a second barcode to nucleic acid molecules in the portion of the single nucleic acid sample that was not in the aliquot;

i) during step d, determining a nucleotide sequence of the first barcode, termed the first nucleotide sequence; and

j) during step e, determining a nucleotide sequence of the second barcode, termed the second nucleotide sequence.

66. (canceled)

69-71. -71. (canceled)