Thermostable type-A DNA polymerase mutants with increased polymerization rate and resistance to inhibitors

• US 9,523,085 B2
• Filed: 08/02/2010
• Issued: 12/20/2016
• Est. Priority Date: 07/31/2009
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Hogrefe, Holly, Cayouette, Michelle, Fox, Jeffrey, Hansen, Connie J., Lapira, Jennifer, Arezi, Bahram, McKinney, Nancy
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US12/848,765
• Publication Number:
  US 20110027833A1
• Time in Patent Office: 2,332 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

The present invention provides mutants of DNA polymerases having an increased rate of incorporation of nucleotides into nucleic acids undergoing polymerization and having an enhanced resistance to inhibitors of DNA polymerase activity. The mutant polymerases are well suited for fast PCR applications, for PCR amplification of targets in samples that contain inhibitors of wild-type polymerases, and for fast PCR amplification of samples containing DNA polymerase inhibitors. In exemplary embodiments, the mutants are mutants of Taq DNA polymerase.

Claims

1. A mutant thermostable Type-A DNA polymerase consisting of or comprising:

a first mutation at residue 507 of wild-type Taq DNA polymerase, which has the sequence of SEQ ID NO;

4, or at a residue corresponding to residue 507 of wild-type Taq DNA polymerase in another thermostable Type-A DNA polymerase; and

at least one additional mutation at a residue selected from 59, 155, 245, 375, 508, 734, and 749 of wild-type Taq DNA polymerase, or at a corresponding residue in another thermostable Type-A DNA polymerase,

wherein the combination of mutations provides a mutant polymerase that possesses a faster polymerization rate and a higher resistance to polymerization activity inhibitors than the wild-type DNA polymerase from which it is derived.

2. The mutant of claim 1, wherein the mutant DNA polymerase is a mutant Taq DNA polymerase, which comprises an E507K mutation as the first mutation.

3. The mutant of claim 1, which is a mutant Taq DNA polymerase and which comprises mutations at the following residues:

G59, V155, L245, L375, E507, E734, and F749.

4. The mutant of claim 3, which comprises the following mutations:

G59W, V155A, L245M, L375V, E507K, E734G, and F749I.

5. The mutant of claim 1, which is a mutant of a thermostable Type-A DNA polymerase other than Taq DNA polymerase and which comprises mutations at residues corresponding to the following residues of Taq DNA polymerase:

G59, V155, L245, L375, E507, E734, and F749.

6. The mutant of claim 1, which is a mutant Taq DNA polymerase and which comprises mutations at the following residues:

G59, L245, L375, E507, K508, E734, and F749.

7. The mutant of claim 6, which comprises the following mutations:

G59W, L245M, L375V, E507K, K508R, E734G, and F749I.

8. The mutant of claim 1, which is a mutant of a thermostable Type-A DNA polymerase other than Taq DNA polymerase and which comprises mutations at residues corresponding to the following residues of Taq DNA polymerase:

G59, L245, L375, E507, K508, E734, and F749.

9. The mutant of claim 1, which comprises mutations at the following residues of Taq DNA polymerase:

L245, E507, and F749, or at corresponding residues in another thermostable Type-A DNA polymerase.

10. The mutant of claim 1, which comprises mutations at the following residues of Taq DNA polymerase:

L245, L375, E507, E734, and F749, or at corresponding residues in another thermostable Type-A DNA polymerase.

11. The mutant of claim 1, consisting of or comprising the sequence of SEQ ID NO:

12, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

18, SEQ ID NO;

20, SEQ ID NO;

24, SEQ ID NO;

26, SEQ ID NO;

28, SEQ ID NO;

30, SEQ ID NO;

32, SEQ ID NO;

34, SEQ ID NO;

36, or SEQ ID NO;

38, wherein the polymerase possesses a faster polymerization activity than wild-type Taq polymerase and is resistant to inhibitors of wild-type Taq polymerase.

12. A kit for amplification of a target nucleic acid, said kit comprising the mutant DNA polymerase of claim 1 and packaging materials therefor.

13. A method of polymerization of a target nucleic acid from a primer that specifically binds to the target nucleic acid, said method comprising:

combining the primer with the target nucleic acid and the mutant thermostable Type-A DNA polymerase of claim 1,

wherein the mutant thermostable Type-A DNA polymerase consists of or comprises;

a first mutation at residue 507 of wild-type Taq DNA polymerase or at a residue corresponding to residue 507 of wild-type Taq DNA polymerase in another thermostable Type-A DNA polymerase; and

at least one additional mutation at a residue selected from 59, 155, 245, 375, 508, 734, and 749 of wild-type Taq DNA polymerase, or at a corresponding residue in another thermostable Type-A DNA polymerase,

wherein the combination of mutations provides a mutant polymerase that possesses a faster polymerization rate and a higher resistance to polymerization activity inhibitors than the wild-type DNA polymerase from which it is derived, and

providing conditions under which the polymerase extends the primer using the sequence of the target as a template for incorporation of nucleotides.

14. The method of claim 13, which is a method of PCR.

15. The method of claim 14, which is a method of fast PCR.

16. The method of claim 13, wherein the conditions include the presence of an inhibitor of the wild-type DNA polymerase at a concentration that is inhibitory to the wild-type DNA polymerase.

17. The method of claim 13, wherein the target nucleic acid is present in blood or a fraction of blood.

18. The method of claim 13, wherein the target nucleic acid is present in plant material or a sample containing plant material.