Nucleic acid sequencing methods and systems

Patent metadata

Field	Value
Publication number	US-10077470-B2
Application number	US-201514805381-A
Country	US
Kind code	B2
Filing date	Jul 21, 2015
Priority date	Jul 21, 2015
Publication date	Sep 18, 2018
Grant date	Sep 18, 2018

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
(a) contacting a primed template nucleic acid with a polymerase that is attached to a label moiety 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;

(b) detecting the ternary complex while precluding incorporation of the test nucleotide into the primer, wherein the detecting comprises detecting the label moiety that is attached to the polymerase; and
(c) identifying the next base of the primed template nucleic acid from the detected ternary complex.

2. The method of claim 1 , wherein the conditions comprise presence of salt at a concentration in the range of 50 to 1500 mM.

3. The method of claim 1 , wherein the conditions comprise presence of potassium glutamate.

4. The method of claim 1 , wherein the conditions comprise presence of a ternary complex stabilizing agent.

5. The method of claim 4 , wherein the ternary complex stabilizing agent comprises a non-catalytic metal ion.

6. The method of claim 5 , wherein the non-catalytic metal ion is strontium, tin, or nickel.

7. The method of claim 1 , further comprising a wash step before step (b), wherein the wash step separates unbound polymerase and unbound nucleotides from the ternary complex, and wherein the wash step occurs under conditions that stabilize the ternary complex.

8. The method of claim 7 , wherein the wash step comprises contacting the ternary complex with a ternary complex stabilizing agent.

9. The method of claim 8 , wherein the ternary complex stabilizing agent is a non-catalytic metal ion.

10. The method of claim 9 , wherein the non-catalytic metal ion is strontium, tin, or nickel.

11. The method of claim 7 , wherein the wash step removes any binary complexes between the primed template nucleic acid and the polymerase.

12. The method of claim 1 , wherein the test nucleotide is unlabeled.

13. A method of identifying a base in a primed template nucleic acid, comprising
(a) contacting a 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 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;

(b) detecting the ternary complex while precluding incorporation of the test nucleotide into the primer, wherein the detecting is carried out in the absence of label moieties on the test nucleotide and on the polymerase; and
(c) identifying the next base of the primed template nucleic acid from the detected ternary complex.

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

15. The method of claim 1 , further comprising an incorporation step following step (b), the incorporation step comprising incorporating into the primer a nucleotide that is complimentary to the next base.

16. The method of claim 15 , wherein steps (a) and (b) are repeated at least once using a different type of test nucleotide prior to the incorporation step.

17. The method of claim 15 , wherein the nucleotide that is incorporated comprises a reversible terminator moiety.

18. The method of claim 17 , wherein the nucleotide that is incorporated is unlabeled.

19. The method of claim 17 , further comprising (d) removing the reversible terminator after step (b).

20. The method of claim 19 , further comprising repeating steps (a) through (d) at least once.

21. The method of claim 1 , further comprising a reloading step following step (b), the reloading step comprising contacting the primed template nucleic acid with a polymerase and the first test nucleotide molecule under the conditions.

22. The method of claim 21 , further comprising an incorporation step following the reloading step, the incorporation step comprising incorporating into the primer a nucleotide that is complimentary to the next base.

23. The method of claim 1 , further comprising repeating steps (a) through (c) at least once.

24. The method of claim 23 , wherein steps (a) through (c) are repeated using a second test nucleotide that is different from the test nucleotide.

25. The method of claim 24 , wherein steps (a) through (c) are repeated using a third test nucleotide that is different from the test nucleotide and the second test nucleotide.

26. The method of claim 25 , wherein steps (a) through (c) are repeated using a fourth test nucleotide that is different from the test nucleotide, the second test nucleotide and the third test nucleotide.

27. The method of claim 13 , wherein detecting step (b) is performed on a surface plasmon resonance sensor.

28. The method of claim 27 , wherein the primed template nucleic acid is located on the surface plasmon resonance sensor.

29. The method of claim 13 , wherein detecting step (b) is performed through detection of an intrinsic signal from the polymerase.

30. The method of claim 29 , wherein the intrinsic signal from the polymerase is a light scattering signal.

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

32. The method of claim 31 , wherein the surface is attached to a clonally amplified population of the primed template nucleic acid and wherein the clonally amplified population is subjected to steps (a) through (c).

33. The method of claim 31 , wherein the surface comprises a plurality of different template nucleic acids that are subjected to steps (a) through (c).

34. The method of claim 1 , wherein the conditions comprise presence of a polymerase inhibitor.

35. 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.

36. The method of claim 1 , wherein the conditions comprise a moiety on the nucleotide that precludes incorporation of the test nucleotide into the primer.

37. The method of claim 1 , further comprising an incorporation step following step (b), the incorporation step comprising replacing the test nucleotide with a second nucleotide and incorporating the second nucleotide into the primer.

38. The method of claim 37 , wherein the second nucleotide is unlabeled.

39. The method of claim 38 , wherein the nucleotide that is incorporated comprises a reversible terminator moiety.

40. The method of claim 39 , further comprising (d) removing the reversible terminator after ste

Assignees

• Omniome 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