POLYMERASES ENGINEERED TO REDUCE NUCLEOTIDE-INDEPENDENT DNA BINDING

• US 20200131486A1
• Filed: 01/09/2020
• Published: 04/30/2020
• Est. Priority Date: 04/29/2016
• Status: Active Application

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Omniome, Inc.
• Inventors: IYIDOGAN, Pinar, WALLEN, Mark C., LIU, Ying L., VIJAYAN, Kandaswamy
• Application Number:
  US16/738,577
• Publication Number:
  US 20200131486A1
• Time in Patent Office: Days
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase

Abstract

Provided are engineered DNA polymerases exhibiting modified functionality, and polynucleotides encoding same. Modified features include: (1) reduced catalytic activity in the presence of magnesium ions and/or (2) reduced affinity for primed template nucleic acid molecules in the absence of cognate nucleotide, and an ability to discriminate between cognate and non-cognate nucleotides under low salt conditions. Sequencing By Binding™ procedures employing the engineered polymerases have certain advantages. The engineered polymerases can have other uses as well.

Claims

1. A method of detecting a next correct nucleotide of 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 and a test nucleotide,

wherein the test nucleotide is the next correct nucleotide of the primed template nucleic acid,

wherein a ternary complex is formed, the ternary complex comprising the primed template nucleic acid, the crippled DNA polymerase and the test nucleotide, and

wherein the crippled DNA polymerase comprises a variant of the sequence of SEQ ID NO;

3, said variant being at least 80% identical to SEQ ID NO;

3 and comprising an amino acid substitution mutation at one or more of positions K250, Q281, D355, Q425, and D532; and

(b) detecting the ternary complex without chemical incorporation of the test nucleotide into the primer of the primed template nucleic acid, thereby detecting the next correct nucleotide.

2. The method of claim 1, wherein the primer of the primed template nucleic acid comprises a reversible terminator nucleotide at its 3′

-end.

3. The method of claim 1, further comprising (c) determining from the results of step (b) the identity of the next template nucleotide of the primed template nucleic acid.

4. The method of claim 1, wherein the variant is at least 90% identical to SEQ ID NO:

3.

5. The method of claim 1,

wherein the substitution mutation at position K250 comprises a mutation to a polar amino acid,

wherein the substitution mutation at position Q281 comprises a mutation to an acidic amino acid,

wherein the substitution mutation at position D355 comprises a mutation to a different acidic amino acid,

wherein the substitution mutation at position Q425 comprises a mutation to a different polar amino acid, and

wherein the substitution mutation at position D532 comprises a mutation to a different acidic amino acid.

6. The method of claim 5,

wherein the substitution mutation at position K250 comprises a mutation to Cys,

wherein the substitution mutation at position Q281 comprises a mutation to Glu,

wherein the substitution mutation at position D355 comprises a mutation to Glu,

wherein the substitution mutation at position Q425 comprises a mutation to Cys, and

wherein the substitution mutation at position D532 comprises a mutation to Glu.

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

8. The method of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E).

9. The method of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).

10. The method of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E), a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).

11. The method of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position D355 that replaces Asp (D) with Glu (E), and a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E).

12. The method of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position D355 that replaces Asp (D) with Glu (E), a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).

13. The method of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position D355 that replaces Asp (D) with Glu (E), a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E), a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).

14. The method of claim 1, wherein the crippled polymerase comprises an exogenous label.

15. A method of detecting a next correct nucleotide of 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 and a test nucleotide,

wherein the test nucleotide is the next correct nucleotide of the primed template nucleic acid,

wherein a ternary complex is formed, the ternary complex comprising the primed template nucleic acid, the crippled DNA polymerase and the test nucleotide, and

wherein the crippled DNA polymerase comprises a variant of the sequence of SEQ ID NO;

2, said variant being at least 80% identical to SEQ ID NO;

2 and comprising an amino acid substitution mutation at one or more of positions Q290, K259 and Q434; and

(b) detecting the ternary complex without chemical incorporation of the test nucleotide into the primer of the primed template nucleic acid, thereby detecting the next correct nucleotide.

16. The method of claim 15, wherein the substitution mutation comprises Glu (E) at position 290.

17. The method of claim 15, wherein the substitution mutation comprises Cys (C) at position 259, and Cys (C) at position 434.

18. The method of claim 15, wherein the substitution mutation comprises Glu (E) at position 290, Cys (C) at position 259, and Cys (C) at position 434.

19. A method of detecting a next correct nucleotide of 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 and a test nucleotide,

wherein the test nucleotide is the next correct nucleotide of the primed template nucleic acid,

wherein a ternary complex is formed, the ternary complex comprising the primed template nucleic acid, the crippled DNA polymerase and the test nucleotide, and

wherein the crippled DNA polymerase comprises a variant of the sequence of SEQ ID NO;

1, said variant being at least 80% identical to SEQ ID NO;

1 and comprising an amino acid substitution mutation at one or more of positions Q307, K276 and Q451; and

(b) detecting the ternary complex without chemical incorporation of the test nucleotide into the primer of the primed template nucleic acid, thereby detecting the next correct nucleotide.

20. The method of claim 19, wherein the substitution mutation comprises Glu (E) at position 307.

21. The method of claim 19, wherein the substitution mutation comprises Cys (C) at position 276, and Cys (C) at position 451.

22. The method of claim 19, wherein the substitution mutation comprises Glu (E) at position 307, Cys (C) at position 276, and Cys (C) at position 451.