Methods for identifying modified bases in nucleic acid templates

Patent metadata

Field	Value
Publication number	US-12527665-B2
Application number	US-202017003388-A
Country	US
Kind code	B2
Filing date	Aug 26, 2020
Priority date	Dec 11, 2008
Publication date	Jan 20, 2026
Grant date	Jan 20, 2026

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 identifying one or more modified bases in a nucleic acid template, the method comprising:
a) providing a circular nucleic acid template that may have one or more modified bases; b) providing a polymerase enzyme having strand displacement activity and capable of processing the circular nucleic acid template; c) contacting the circular nucleic acid template with the polymerase enzyme; d) monitoring processing of the circular nucleic acid template by the polymerase enzyme, wherein the polymerase enzyme makes multiple passes around the circular nucleic acid template, whereby the processing results in the synthesis of a nascent nucleic acid strand, and wherein the monitoring: (i) occurs during the processing, and (ii) detects incorporation of single nucleotides into the nascent nucleic acid strand, thereby generating multiple sequence reads complementary to the circular nucleic acid template; e) measuring the kinetics of single nucleotide incorporations; and f) identifying, if present, one or more modified bases within the circular nucleic acid template using the measured kinetics of nucleotide incorporations for the multiple sequence reads.

2 . The method of claim 1 , wherein the identifying includes using kinetic measurements for nucleotides incorporated upstream and downstream of the one or more modified bases in the nucleic acid template.

3 . The method of claim 1 , wherein the kinetic measurements include interpulse duration (IPD) or pulse width (PW).

4 . The method of claim 1 , wherein the kinetic measurements include interpulse duration (IPD) and pulse width (PW).

5 . The method of claim 1 , wherein the kinetic measurements from the multiple sequence reads are averaged.

6 . The method of claim 1 , wherein the polymerase makes 3 or more passes around the circular nucleic acid template.

7 . The method of claim 1 , wherein the polymerase makes 5 or more passes around the circular nucleic acid template.

8 . The method of claim 1 , wherein the circular nucleic acid template comprises a double-stranded nucleic acid capped with hairpins.

9 . The method of claim 8 , wherein the hairpins comprise barcodes.

10 . The method of claim 1 , wherein the one or more modified bases include a methylated cytosine or a methylated adenine base.

11 . The method of claim 1 , wherein the one or more modified bases include 5-methyl cytosine.

12 . The method of claim 1 , wherein the one or more modified bases include 5-hydroxymethyl cytosine.

13 . The method of claim 1 , wherein the circular nucleic acid template comprises DNA.

14 . The method of claim 1 , wherein the circular nucleic acid template is DNA.

15 . The method of claim 1 , wherein prior to providing the circular nucleic acid template, the nucleic acid template is subjected to a treatment to alter a modified base of the one or more modified bases.

16 . The method of claim 15 , wherein the treatment is selected from the group consisting of: glycosylase modification, DMS modification, cytosine methyltransferase modification, TET 1 modification, restriction enzyme modification, and cytidine deaminase modification.

17 . The method of claim 1 , wherein the polymerase enzyme is immobilized at optically resolvable reaction sites on a substrate during the monitoring.

18 . The method of claim 1 , wherein the circular nucleic acid template comprises regions of internal complementarity.

19 . The method of claim 18 , wherein the regions of internal complementarity are used to map positions of modified bases in the nucleic acid template.

Assignees

• Pacific Biosciences California Inc

Inventors

• Flusberg Benjamin
• Turner Stephen
• Sorenson Jon
• Travers Kevin
• Heiner Cheryl

Classifications

• G16B30/00

  ICT specially adapted for sequence analysis involving nucleotides or amino acids · CPC title

• C12Q1/6869

  Methods for sequencing · CPC title

• A61F2250/0036

  differing in thickness · CPC title

• A61F2250/0014

  having different values of a given property or geometrical feature, e.g. mechanical property or material property, at different locations within the same prosthesis · CPC title

• A61F2230/0071

  spherical · CPC title