DNA polymerases

• US 10,590,399 B2
• Filed: 09/29/2016
• Issued: 03/17/2020
• Est. Priority Date: 08/03/2011
• Status: Active Grant

• Current Assignee: Thermo Fisher Scientific Baltics, UAB (Thermo Fisher Scientific Incorporated)
• Original Assignee: Thermo Fisher Scientific Baltics, UAB (Thermo Fisher Scientific Incorporated)
• Inventors: Skirgaila, Remigijus, Tubelevic̆iute, Agne, Rims̆eliene, Renata, Burinskas, Sigitas
• Primary Examiner(s): Mondesi, Robert B
• Assistant Examiner(s): Epstein, Todd M
• Application Number:
  US15/280,077
• Publication Number:
  US 20170081646A1
• Time in Patent Office: 1,265 Days
• Field of Search: None
• CPC Class Codes: C07K 2319/21 containing a His-tag
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

A DNA polymerase mutant comprising a Taq DNA polymerase amino acid sequence with a mutation at one or more of the following selected amino acid positions: E189K, E230K, E507K, H28R, L30R, G38R, F73V, H75R, E76A, E76G, E76K, E90K, K206R, E315K, A348V, L351F, A439T, D452N, G504S, E507A, D551N, L552R, I553V, D578N, H676R, Q680R, D732G, E734G, E734K, F749V; wherein the polymerase mutant exhibits relative to wild-type DNA polymerase increased polymerase speed, increased affinity to DNA substrate and/or increased resistance to a DNA polymerase inhibitor; and wherein, when the mutation is E507K in combination with two or more further mutations or the mutation is Q680R in combination with four or more further mutations, at least one of the further mutations is at one of the selected amino acid positions; and when the mutation is I553V, this is not in combination with D551S.

Claims

1. A Taq DNA polymerase mutant comprising SEQ ID NO:

13 amino acid sequence with a mutation at position E230 and optionally one or more of the following mutations;

E189K, H28R, L30R, G38R, F73V, H75R, E76A, E76G, E76K, E90K, K206R, E315K, A348V, L351F, A439T, D452N, G504S, E507A, E507K, D551N, L552R, I553V, D578N, H676R, Q680R, D732G, E734G, E734K, and F749V;

wherein the polymerase mutant exhibits relative to wild-type DNA polymerase increased polymerase speed, increased affinity to DNA substrate and/or increased resistance to a DNA polymerase inhibitor.

2. A DNA polymerase mutant according to claim 1, wherein the amino acid sequence has a mutation at no more than three amino acid positions.

3. A DNA polymerase mutant according to claim 2, wherein the amino acid sequence has a mutation at only one amino acid position.

4. A DNA polymerase mutant according to claim 2, wherein the amino acid sequence has a mutation at only two amino acid positions.

5. A DNA polymerase mutant according to claim 1, which exhibits increased polymerase speed relative to wild-type DNA polymerase.

6. A DNA polymerase mutant according to claim 5, wherein the Taq DNA polymerase amino acid sequence comprises one or more of the following mutations:

E189K, E230K, E507K, H28R, L30R, F73V, H75R, E76A, E76G, E76K, E90K, K206R, A439T, D452N, G504S, D551N, I553V, H676R, D732G, E734G, F749V.

7. A DNA polymerase mutant according to claim 5, which exhibits an increased polymerase speed which is at least 1.5 times faster than wild-type DNA polymerase.

8. A DNA polymerase mutant according to claim 5, which exhibits an increased polymerase speed which is at least 3 times faster than wild-type DNA polymerase.

9. A DNA polymerase mutant according to claim 5, which exhibits an increased polymerase speed which is at least 12 times faster than wild-type DNA polymerase.

10. A DNA polymerase mutant according to claim 1, which exhibits increased affinity to DNA substrate relative to wild-type DNA polymerase.

11. A DNA polymerase mutant according to claim 10, wherein the Taq DNA polymerase amino acid sequence comprises one or more of the following mutations:

E189K, E230K, E507K, H75R, E315K, A348V, L351F, L552R, D578N.

12. A DNA polymerase mutant according to claim 10, wherein the Kd for a DNA oligoduplex substrate is no more than 1 nM, as measured by electrophoretic shift mobility assay following incubation in 40 mM Tris, 20 mM acetic acid, 1 mM EDTA at pH8.4, in the presence of 10% v/v glycerol at 4°

C. for 30 mins.

13. A DNA polymerase mutant according to claim 1, which exhibits increased resistance to a DNA polymerase inhibitor selected from SYBR Green I dye, blood, SDS, guanidinium salts and heparin.

14. A DNA polymerase mutant according to claim 13, wherein the Taq DNA polymerase amino acid sequence comprises one or more of the following mutations:

E189K, E230K, E507K, H28R, L30R, G38R, H75R, E76A, E76G, E76K, E90K, E315K, A439T, D452N, G504S, E507A, D551N, L552R, I553V, D578N, H676R, Q680R, D732G, E734G, E734K.

15. A DNA polymerase mutant according to claim 13, wherein the Kd for a DNA oligoduplex substrate in the presence of 0.4 μ

M SYBR Green I dye is no more than 10 nM, as measured by electrophoretic shift mobility assay following incubation in 40 mM Tris, 20 mM acetic acid, 1 mM EDTA at pH8.4, in the presence of 10% v/v glycerol at 4°

C. for 30 mins.

16. A DNA polymerase mutant according to claim 1, wherein the amino acid sequence has a mutation at only two amino acid positions;

the DNA polymerase mutant exhibits increased affinity to DNA substrate relative to wild-type DNA polymerase; and

/or the DNA polymerase mutant exhibits increased resistance to a DNA polymerase inhibitor selected from SYBR Green I dye, blood, SDS, guanidinium salts and heparin; and

wherein the Taq DNA polymerase amino acid sequence comprises the following mutations;

E230K+E507K or E230K+E315K.

17. A DNA polymerase mutant according to claim 1, wherein the Taq DNA polymerase amino acid sequence comprises one or more of the following mutations:

E189K, E230K and E507K.

18. A DNA polymerase mutant according to claim 1, wherein the Taq DNA polymerase amino acid sequence comprises an E230K mutation.

19. A DNA polymerase mutant according to claim 1, wherein the Taq DNA polymerase amino acid sequence comprises an E507K mutation.

20. A kit for nucleic acid amplification, which comprises a DNA polymerase mutant according to claim 1 and one or more reagents for a DNA synthesis reaction.

21. A DNA polymerase obtainable by a process comprising:

(1) subjecting a polynucleotide encoding a Taq DNA polymerase to error-prone PCR to generate a library comprising an array of different polynucleotides;

(2) screening the library for increased polymerase speed, increased polymerase affinity to DNA substrate or increase resistance to a DNA polymerase inhibitor;

(3) selecting one or more DNA polymerases from screening step 2; and

(4) repeating steps 1 to 3 until a final DNA polymerase is obtained;

wherein the final DNA polymerase comprises SEQ ID NO;

13 amino acid sequence with a mutation at position E230, and optionally one or more of the following mutations;

E189K, H28R, L30R, G38R, F73V, H75R, E76A, E76G, E76K, E90K, K206R, E315K, A348V, L351F, A439T, D452N, G504S, E507A, E507K, D551N, L552R, I553V, D578N, H676R, Q680R, D732G, E734G, E734K, and F749V.