Method of nucleic acid sequence determination

• US 10,584,379 B2
• Filed: 04/28/2017
• Issued: 03/10/2020
• Est. Priority Date: 04/29/2016
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Omniome, Inc.
• Inventors: Vijayan, Kandaswamy, Iyidogan, Pinar
• Primary Examiner(s): Mummert, Stephanie K
• Application Number:
  US15/581,822
• Publication Number:
  US 20170314072A1
• Time in Patent Office: 1,047 Days
• Field of Search: None
• CPC Class Codes: C12N 9/1247 DNA-directed RNA polymerase (2.7.7.6)
• 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 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Y 207/07006 DNA-directed RNA polymerase (2.7.7.6)

Abstract

Provided are sequencing-by-binding methods of detecting cognate nucleotides using a crippled DNA polymerizing enzyme that possesses the ability to bind the next correct nucleotide downstream of a primer in a template-dependent fashion, but does not possess the activity needed to promote phosphodiester bond formation. Use of the crippled DNA polymerase permits interrogation of one nucleotide at a time, without incorporation of any nucleotide. Labeled nucleotides, such as fluorescently labeled nucleotides, can be used in conjunction with the crippled DNA polymerase to establish cognate nucleotide identity in a rapid manner.

Claims

1. A method of determining whether a test nucleotide is the next correct nucleotide comprising a base complementary to the next base in a template strand immediately downstream of a primer in a primed template nucleic acid, comprising the steps of:

(a) contacting the primed template nucleic acid with a first reaction mixture that comprises a crippled DNA polymerase comprising either a polypeptide sequence comprising SEQ ID NO;

12 or a polypeptide sequence comprising SEQ ID NO;

14 and the test nucleotide,

whereby, if the test nucleotide is the next correct nucleotide, there is formed a complex comprising the primed template nucleic acid, the crippled DNA polymerase and the test nucleotide, and

wherein the crippled DNA polymerase is substantially incapable of magnesium-catalyzed phosphodiester bond formation;

(b) measuring binding of the primed template nucleic acid to the crippled DNA polymerase in the presence of the test nucleotide, without chemical incorporation of the test nucleotide into the primer of the primed template nucleic acid; and

(c) determining from the results of step (b) whether the test nucleotide is the next correct nucleotide.

2. The method of claim 1, wherein the crippled DNA polymerase catalyzes formation of phosphodiester bonds in the presence of divalent manganese ions, and wherein the first reaction mixture does not contain a concentration of divalent manganese ions that promotes formation of phosphodiester bonds.

3. The method of claim 1, wherein the test nucleotide comprises an exogenous label.

4. The method of claim 2, wherein the exogenous label of the test nucleotide comprises a fluorescent moiety, and wherein step (b) comprises measuring a fluorescent signal produced by the fluorescent moiety of the test nucleotide.

5. The method of claim 1, wherein the crippled DNA polymerase comprises an exogenous label, and wherein step (b) comprises detecting the exogenous label of the crippled DNA polymerase.

6. The method of claim 5, wherein the exogenous label of the crippled DNA polymerase comprises a fluorescent moiety, and wherein step (b) comprises measuring a fluorescent signal produced by the fluorescent moiety of the crippled DNA polymerase.

7. The method of claim 1, wherein the primer comprises a free 3′

hydroxyl moiety.

8. The method of claim 1, further comprising, after step (b), the step of replacing the first reaction mixture with a second reaction mixture that comprises a second polymerase and a second type of nucleotide, and then incorporating the second type of nucleotide into the primer of the primed template nucleic acid.

9. The method of claim 8, wherein the second type of nucleotide is a reversible terminator nucleotide that comprises a reversible terminator moiety, and wherein incorporation of the reversible terminator nucleotide produces a blocked primed template nucleic acid molecule.

10. The method of claim 9, further comprising the step of removing the reversible terminator moiety from the blocked primed template nuclei acid molecule to regenerate the primed template nucleic acid molecule.

11. The method of claim 9, further comprising repeating steps (a)-(c) using the blocked primed template nucleic acid molecule in place of the primed template nucleic acid.

12. The method of claim 10, further comprising repeating steps (a)-(c).

13. The method of claim 1, wherein the polypeptide sequence of the crippled DNA polymerase is either SEQ ID NO:

1 with the exception of comprising SEQ ID NO;

12, or SEQ ID NO;

1 with the exception of comprising SEQ ID NO;

14.

14. The method of claim 1, wherein the polypeptide sequence of the crippled DNA polymerase is either SEQ ID NO:

2 with the exception of comprising SEQ ID NO;

12, or SEQ ID NO;

2 with the exception of comprising SEQ ID NO;

14.

15. The method of claim 1, wherein the polypeptide sequence of the crippled DNA polymerase is either SEQ ID NO:

3 with the exception of comprising SEQ ID NO;

12, or SEQ ID NO;

3 with the exception of comprising SEQ ID NO;

14.

16. The method of claim 1, wherein the first reaction mixture comprises divalent magnesium ion.

17. The method of claim 9, wherein the first reaction mixture comprises Mg²⁺ ions, and wherein the primer comprises a 3′

hydroxyl moiety.