Method and system for sequencing nucleic acids

• US 10,443,098 B2
• Filed: 08/15/2017
• Issued: 10/15/2019
• Est. Priority Date: 08/15/2016
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Omniome, Inc.
• Inventors: Vijayan, Kandaswamy, Dambacher, Corey M., Tu, Eugene, Bernard, Mark A., Rokicki, Joseph, Wilson, Kerry
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US15/677,870
• Publication Number:
  US 20180044727A1
• Time in Patent Office: 791 Days
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2565/133 conformational analysis
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: G01N 33/50 Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q

Abstract

Provided are compositions, methods and systems for determining the sequence of a template nucleic acid using a polymerase-based, sequencing-by-binding procedure. An examination step involves monitoring the interaction between a polymerase and template nucleic acid in the presence of one or more nucleotides. Identity of the next correct nucleotide in the sequence is determined without incorporation of any nucleotide into the structure of the primer by formation of a phosphodiester bond. An optional incorporation step can be used after the examination step to extend the primer by one or more nucleotides, thereby incrementing the template nucleotides that can be examined in a subsequent examination step. The sequencing-by-binding procedure does not require the use of labeled nucleotides or polymerases, but optionally can employ these reagents.

Claims

1. A method of identifying a template base in a primed template nucleic acid, the method comprising:

(a) contacting a primed template nucleic acid with a polymerase and nucleotides that complement four different types of bases in the template nucleic acid, under conditions that stabilize a first ternary complex at a template base, the first ternary complex comprising the primed template nucleic acid, polymerase and a cognate nucleotide for the template base;

(b) detecting the first ternary complex while precluding incorporation of any nucleotide into the primer;

(c) removing the polymerase and the cognate nucleotide from the primed template nucleic acid;

(d) contacting the primed template nucleic acid from step (c) with a polymerase and nucleotides that complement four different types of bases in the template nucleic acid, under conditions that stabilize a second ternary complex at the template base, the second ternary complex comprising the primed template nucleic acid, polymerase and a cognate for the template base nucleotide;

(e) detecting the second ternary complex while precluding incorporation of any nucleotide into the primer; and

(f) identifying the template base from the first ternary complex and the second ternary complex.

2. The method of claim 1, wherein the first and second ternary complexes are stabilized by the presence of a reversible terminator moiety on the 3′

terminal nucleotide of the primer.

3. The method of claim 2, further comprising, after step (d), removing the reversible terminator moiety on the 3′

terminal nucleotide of the primer.

4. The method of claim 1, wherein each of the cognate nucleotides is an unlabeled nucleotide.

5. The method of claim 1, wherein the polymerases comprise exogenous labels that are detected.

6. The method of claim 1, wherein the cognate nucleotides comprise exogenous labels that are detected.

7. The method of claim 1, further comprising (g) incorporating a nucleotide at the 3′

end of the primer after step (e), thereby producing an extended primed template nucleic acid.

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

9. The method of claim 8, wherein the nucleotide that is incorporated is an unlabeled reversible terminator nucleotide.

10. The method of claim 7, further comprising repeating steps (a) through (g) using the extended primed template nucleic acid in place of the primed template nucleic acid, thereby sequencing the template nucleic acid.

11. A method of sequencing a template nucleic acid, comprising:

(a) contacting a primed template nucleic acid with a polymerase and a first combination of nucleotides that complement four different types of bases in the template nucleic acid, under conditions that form a stabilized ternary complex between the polymerase, primed template nucleic acid and a nucleotide that is complementary to a next base of the primed template nucleic acid;

(b) detecting the ternary complex while precluding incorporation of nucleotides into the primer;

(c) repeating steps (a) and (b) using the primed template nucleic acid, a polymerase and a second combination of nucleotides that complement four different types of bases in the template nucleic acid, thereby forming and examining a second ternary complex at the next base of the primed template nucleic acid;

(d) incorporating into the primer, after step (c), a nucleotide that is complementary to the next base, thereby producing an extended primed template nucleic acid; and

(e) repeating steps (a) through (d) using the extended primed template nucleic acid in place of the primed template nucleic acid, thereby sequencing the primed template nucleic acid.

12. The method of claim 11, further comprising removing the polymerase and next correct nucleotide from the primed template nucleic acid prior to repeating steps (a) and (b).

13. The method of claim 11, wherein the ternary complex is stabilized by the presence of a reversible terminator moiety on the 3′

terminal nucleotide of the primer.

14. The method of claim 13, further comprising, after step (c), removing the reversible terminator moiety on the 3′

terminal nucleotide of the primer.

15. The method of claim 11, wherein the nucleotide that is complementary to the next base of the primed template nucleic acid is an unlabeled nucleotide.

16. The method of claim 11, wherein the polymerase comprises an exogenous label that is detected in step (b).

17. The method of claim 11, wherein the nucleotide that is complementary to the next base of the primed template nucleic acid comprises an exogenous label that is detected in step (b).

18. The method of claim 11, wherein the nucleotide that is incorporated into the primer is an unlabeled nucleotide.

19. The method of claim 18, wherein the nucleotide that is incorporated into the primer is an unlabeled reversible terminator nucleotide.