Nucleic acid sequencing methods and systems

Patent metadata

Field	Value
Publication number	US-11499185-B2
Application number	US-201816046707-A
Country	US
Kind code	B2
Filing date	Jul 26, 2018
Priority date	Jul 21, 2015
Publication date	Nov 15, 2022
Grant date	Nov 15, 2022

Abstract

The present disclosure provides compositions, methods and systems for sequencing a template nucleic acid using a polymerase based, nucleic acid binding reaction involving examination of the interaction between a polymerase and template nucleic acid in the presence of one or more unlabeled nucleotides. The methods rely, in part, on identifying a base of a template nucleic acid during nucleic acid synthesis by controlling the sequencing reaction conditions. Template nucleic acid bases may be identified during an examination step followed by an optional incorporation step.

First claim

What is claimed is:

1. A method of identifying a base in a primed template nucleic acid, comprising
I. performing an examination step comprising:
(a) providing a primed template nucleic acid comprising a primer, wherein the primer comprises a reversible terminator moiety at the 3′ end;
(b) contacting the primed template nucleic acid with a polymerase and a test nucleotide, under conditions that:
(i) stabilize ternary complex formed between the polymerase, primed template nucleic acid and test nucleotide when the test nucleotide is complementary to the next base of the primed template nucleic acid while precluding incorporation of the test nucleotide into the primer, and
(ii) destabilize binary complex formed between the primed template nucleic acid and the polymerase when the test nucleotide is not complementary to the next base of the primed template nucleic acid;

(c) washing to remove any unbound polymerase and unbound nucleotides, wherein the washing occurs under conditions that stabilize the ternary complex;
(d) detecting the ternary complex while precluding incorporation of the test nucleotide into the primer;
(e) identifying the next base of the primed template nucleic acid from the detected ternary complex.

2. The method of claim 1 , wherein the washing comprises contacting the ternary complex with a ternary complex stabilizing agent.

3. The method of claim 1 , further comprising a reloading step, the reloading step comprising contacting the primed template nucleic acid with a polymerase and a nucleotide that is complementary to the next base.

4. The method of claim 3 , further comprising an incorporation step following the reloading step, the incorporation step comprising incorporating into the primer a nucleotide that is complementary to the next base.

5. The method of claim 1 , further comprising an incorporation step, the incorporation step comprising incorporating into the primer a nucleotide that is complementary to the next base.

6. The method of claim 5 , wherein step I. is repeated at least once using a different type of test nucleotide prior to the incorporation step.

7. The method of claim 5 , wherein the nucleotide that is incorporated comprises a reversible terminator moiety.

8. The method of claim 7 , wherein the nucleotide that is incorporated is unlabeled.

9. The method of claim 5 , wherein the incorporation step further comprises replacing the polymerase with a different type of polymerase that catalyzes the incorporation.

10. The method of claim 1 , further comprising an incorporation step, the incorporation step comprising replacing the test nucleotide with a second nucleotide and incorporating the second nucleotide into the primer.

11. The method of claim 10 , wherein the second nucleotide comprises a reversible terminator moiety.

12. The method of claim 11 , wherein the second nucleotide is unlabeled.

13. The method of claim 10 , wherein the incorporation step further comprises replacing the polymerase with a different type of polymerase that catalyzes the incorporation.

14. The method of claim 1 , wherein the conditions comprise presence of a polymerase inhibitor that precludes incorporation of the test nucleotide into the primer.

15. The method of claim 1 , wherein the conditions comprise an amino acid mutation in the polymerase that precludes incorporation of the test nucleotide into the primer.

16. The method of claim 1 , wherein the conditions comprise presence of a reversible terminator on the 3′ end of the primer that precludes incorporation of the test nucleotide into the primer.

17. The method of claim 1 , wherein the test nucleotide comprises a label moiety.

18. The method of claim 1 , further comprising repeating step I. at least once using a second test nucleotide that is different from the test nucleotide.

19. The method of claim 1 , wherein the primed template nucleic acid is attached to a surface.

20. The method of claim 19 , wherein the surface is attached to a clonally amplified population of the primed template nucleic acid.

21. The method of claim 19 , wherein the surface comprises a plurality of different template nucleic acids.

22. The method of claim 1 , wherein the conditions of step (b) comprise presence of a non-catalytic metal ion.

23. The method of claim 1 , wherein the conditions of step (b) comprise presence of salt at a concentration in the range of 50 to 1500 mM.

24. The method of claim 1 , wherein the detecting comprises detecting a change in refractive index, a light scattering signal or a detectable tag.

Assignees

• Omniome Inc
• Pacific Biosciences California Inc

Inventors

• Vijayan Kandaswamy
• Tu Eugene
• Bernard Mark A

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2563/103

  luminescence · CPC title

• C12Q2565/607

  being a sensor, e.g. electrode · CPC title