Methods for identifying nucleic acid modifications

• US 9,175,338 B2
• Filed: 11/12/2010
• Issued: 11/03/2015
• 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: Flusberg, Benjamin, Korlach, Jonas, Wegener, Jeffrey, Clark, Tyson A., Vilfan, Igor, Kislyuk, Andrey, Turner, Stephen, Sorenson, Jon, Travers, Kevin, Heiner, Cheryl, Tomaney, Austin B., Marks, Patrick, Lee, Jessica, Jia, Lei, Webster, Dale, Lyle, John, Hanes, Jeremiah, Puglisi, Joseph
• Primary Examiner(s): BERTAGNA, ANGELA MARIE
• Application Number:
  US12/945,767
• Publication Number:
  US 20110183320A1
• Time in Patent Office: 1,817 Days
• Field of Search: None
• US Class Current: 1/1
• 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 enhancing identification of a modification of interest, the method comprising:

a) providing a DNA template comprising the modification of interest, the modification of interest being characterized by a first kinetic signature during processing by a polymerase, wherein the modification of interest is a methylated base or a hydroxymethylated base;

b) converting the modification of interest into a further modification, by subjecting the DNA template to a treatment to introduce the further modification, wherein the treatment comprises exposure to a modifying agent selected from the group consisting of a glycosylase, bisulfite, a hydroxylase, a glucosyltransferase, NMIA, and CDI, wherein the further modification is characterized by a second kinetic signature during processing by the polymerase, and further wherein the second kinetic signature is distinct from the first kinetic signature;

c) providing the polymerase, which is capable of processing the DNA template;

d) contacting the DNA template comprising the further modification with the polymerase;

e) monitoring processing of the DNA template comprising the further modification by the polymerase; and

f) detecting a change in the kinetics of the polymerase during the processing, wherein the change is indicative of the second kinetic signature, thereby detecting the further modification, and enhancing identification of the modification of interest that was present in the DNA template prior to the converting.

2. The method of claim 1, wherein the DNA template comprises two single-stranded portions and one double-stranded portion, wherein the two single-stranded portions are single-stranded hairpins.

3. The method of claim 1, wherein the DNA template comprises a first polynucleotide region comprising the modification of interest 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 DNA template.

4. The method of claim 1, wherein the treatment comprises addition of a glucose moiety to a nucleobase comprising the modification of interest, wherein the change in the kinetics of the polymerase during the processing is greater than in the absence of the glucose moiety.

5. The method of claim 4, wherein the nucleobase is a hydroxymethylcytosine nucleobase, which is converted to β

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

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

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

8. The method of claim 6, wherein the monitoring detects incorporation of single nucleotides into the nascent nucleic acid strand to generate a sequence read that is complementary to the DNA template.

9. The method of claim 8, wherein the change occurs at the further modification and at one or more positions upstream or downstream of the further modification.

10. The method of claim 8, further comprising mapping the modification of interest within the DNA template, the mapping comprising:

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

b) determining the complement of the sequence complementary to the DNA template in f; and

c) mapping the modification of interest at a position in the DNA template that is proximal to the complement of the sequence complementary to the DNA template in f.

11. The method of claim 1, wherein the DNA template and the polymerase form a complex that is immobilized at a reaction site on a substrate.

12. The method of claim 1, wherein the DNA template is a plurality of DNA templates that are optically resolvable from one another during the monitoring.

13. The method of claim 1, wherein the second kinetic signature comprises kinetic changes in the polymerase at one or more positions upstream or downstream of the further modification.

14. The method of claim 1, wherein the DNA template further comprises a third modification, wherein the third modification is:

(i) different from both the modification of interest and the further modification; and

(ii) is characterized by a third kinetic signature during processing by the polymerase, wherein the third kinetic signature is distinct from both the first kinetic signature and the second kinetic signature;

and further wherein said detecting comprises distinguishing between the further modification and the third modification during the processing based upon their kinetic signatures.

15. The method of claim 4, wherein the glucose moiety comprises a detectable label.