Nucleic acid sequence analysis

Patent metadata

Field	Value
Publication number	US-9551028-B2
Application number	US-201414552901-A
Country	US
Kind code	B2
Filing date	Nov 25, 2014
Priority date	Sep 19, 2008
Publication date	Jan 24, 2017
Grant date	Jan 24, 2017

Abstract

Provided are methods for sequencing a nucleic acid with a sequencing enzyme, e.g., a polymerase or exonuclease. The sequencing enzyme can optionally be exchanged with a second sequencing enzyme, which continues the sequencing of the nucleic acid. In certain embodiments, a template is fixed to a surface through a template localizing moiety. The template localizing moiety can optionally anneal with the nucleic acid and/or associate with the sequencing enzyme. Also provided are compositions comprising a nucleic acid and a first sequencing enzyme, which can sequence the nucleic acid and optionally exchange with a second sequencing enzyme present in the composition. Compositions in which a template localizing moiety is immobilized on a surface are provided. Also provided are methods for using data from analytical reactions wherein two different enzymes are employed, e.g., at a same or different reaction regions.

First claim

What is claimed is:

1. A method of sequencing a single template molecule, the method comprising:
localizing the single template molecule to a reaction region through association with a template localizing moiety attached to the reaction region, wherein the template localizing moiety comprises a protein that translocates along the single template molecule and does not synthesize a polynucleotide complementary to the single template molecule while retaining the single template molecule at the reaction region; and
sequencing a polynucleotide region of the localized single template molecule to obtain a first polynucleotide sequence read of the polynucleotide region, wherein the polynucleotide sequence read comprises redundant sequence information for the polynucleotide region, thereby sequencing the single template molecule.

2. The method of claim 1 , wherein a single strand of the single template nucleic acid can move through the template localizing moiety.

3. The method of claim 1 , wherein the template localizing moiety topologically encircles the single template molecule.

4. The method of claim 1 , wherein the protein is selected from the group consisting of: a helicase, a sliding clamp, a DNA repair enzyme, and a polymerase mutant, fragment, or subunit thereof that does not synthesize a polynucleotide complementary to the single template molecule.

5. The method of claim 1 , wherein the template localizing moiety catalyzes unidirectional, processive strand separation on the single template molecule.

6. The method of claim 1 , wherein the reaction region comprises a zero-mode waveguide.

7. The method of claim 1 , wherein the single template molecule is a circular template molecule.

8. The method of claim 1 , wherein the single template molecule comprises a double-stranded fragment and an end annealed to a stem-loop adapter.

9. The method of claim 1 , wherein the sequencing comprises a method selected from the group consisting of exonuclease sequencing, nanopore-based sequencing, and sequencing by synthesis.

10. The method of claim 1 , wherein the sequencing comprises monitoring the reaction region in real time.

11. A method of sequencing a single nucleic acid molecule, the method comprising:
localizing the single nucleic acid molecule to a reaction region through association with a template localizing moiety attached to the reaction region, wherein the template localizing moiety comprises a protein selected from the group consisting of: a helicase, a sliding clamp, a DNA repair enzyme, and a polymerase mutant, fragment, or subunit thereof that does not synthesize a polynucleotide complementary to the single template molecule; and
sequencing a polynucleotide region of the localized single template nucleic acid molecule using a method selected from the group consisting of exonuclease sequencing, nanopore-based sequencing, and sequencing by synthesis to obtain a polynucleotide sequence read of the polynucleotide region, thereby sequencing the single nucleic acid molecule.

12. The method of claim 11 , wherein the reaction region comprises a zero-mode waveguide.

13. The method of claim 11 , wherein the single nucleic acid molecule is a circular template molecule.

14. The method of claim 11 , wherein the single nucleic acid molecule comprises a double-stranded fragment comprising at least one stem-loop adapter annealed to an end.

15. The method of claim 11 , wherein the sequencing comprises monitoring the reaction region in real time.

16. A method of generating redundant sequence information for a polynucleotide region from multiple template nucleic acid molecules, the method comprising:
providing multiple template nucleic acid molecules, each of which comprises the same polynucleotide region;
localizing each of the template nucleic acid molecules to a different reaction region through association with a template localizing moiety attached to each reaction region, wherein each template localizing moiety encircles each template nucleic acid molecule such that a strand of said template nucleic acid molecule can move through the template localizing moiety, and further wherein the template localizing moiety does not synthesize a polynucleotide complementary to the single template molecule;
sequencing the polynucleotide region in each of the template nucleic acid molecules using a method selected from the group consisting of: exonuclease sequencing, nanopore-based sequencing, and sequencing by synthesis to obtain multiple polynucleotide sequence reads of the polynucleotide region, wherein the polynucleotide sequence reads comprises redundant sequence information for the polynucleotide region, thereby generating redundant sequence information from the template nucleic acid molecules.

17. The method of claim 16 , wherein each template localizing moiety comprises a protein lacking nascent strand synthesis activity that translocates along the template nucleic acid molecules while retaining the template nucleic acid molecules at each different reaction region.

18. The method of claim 16 , wherein each template localizing moiety comprises a protein selected from the group consisting of: a helicase, a sliding clamp, a DNA repair enzyme, and a polymerase mutant, fragment, or subunit thereof that does not synthesize a polynucleotide complementary to the single template molecule.

19. The method of claim 16 , wherein each template localizing moiety catalyzes unidirectional, processive strand separation on each single template molecule.

20. The method of claim 16 , wherein each reaction region comprises a zero-mode waveguide.

21. The method of claim 16 , wherein the sequencing comprises monitoring each of the different reaction regions in real time.

Assignees

• Pacific Biosciences California Inc

Inventors

• Bjornson Keith
• Bibillo Arek
• Christians Fred
• Travers Kevin
• Emig Robin
• Turner Stephen

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C07K14/00

  Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof · CPC title

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2531/125

  Rolling circle · CPC title

• C12Q2521/101

  DNA polymerase · CPC title