Methods for identifying nucleic acid modifications

Patent metadata

Field	Value
Publication number	US-9175338-B2
Application number	US-94576710-A
Country	US
Kind code	B2
Filing date	Nov 12, 2010
Priority date	Dec 11, 2008
Publication date	Nov 3, 2015
Grant date	Nov 3, 2015

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.

First claim

What is claimed is:

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.

Assignees

• 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
• Pacific Biosciences California

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

Classifications

• C12Q2525/117

  incorporating modified base · CPC title

• C12Q2527/113

  Time · CPC title

• C12Q1/6858 Primary

  Allele-specific amplification · CPC title

• C12Q2525/307

  Circular oligonucleotides · CPC title

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title