DNA polymerases

• US 9,493,848 B2
• Filed: 08/02/2012
• Issued: 11/15/2016
• 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: Tubeleviit, Agne, Rimelin, Renata, Skirgaila, Remigijus, Burinskas, Sigitas
• Primary Examiner(s): Mondesi, Robert
• Assistant Examiner(s): Epstein, Todd M
• Application Number:
  US13/564,940
• Publication Number:
  US 20130034879A1
• Time in Patent Office: 1,566 Days
• Field of Search: None
• US Class Current: 1/1
• 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

The invention provides novel mutant thermostable DNA polymerases having desirable properties such as increased polymerase efficiency and speed, increased affinity to DNA substrates and/or increased resistance to DNA polymerase inhibitors. In certain embodiments, the invention provides methods of producing, amplifying and/or sequencing nucleic acid molecules (particularly cDNA molecules) using kits, compositions and/or reactions mixtures containing such novel DNA polymerases.

Claims

1. A DNA polymerase mutant comprising SEQ ID NO:

13 with a mutation at position E189 and optionally one or more of the following mutations;

E230K, E76G, E76K, E76A, E90K, D551N, K206R, Q680R, L552R, H28R, E315K, L30R, L30P, D452N, G504S, G504D, G38R, F73V, H75R, A348V, L351F, A439T, L552R, I553V, D578N, H676R, D732G, E734G, E734K, F749V, E507K, E507A;

wherein the DNA polymerase mutant exhibits relative to wild-type DNA polymerase of SEQ ID NO;

13, and under the same conditions, increased polymerase speed, increased affinity to DNA substrate and/or increased resistance to a DNA polymerase inhibitor.

2. The DNA polymerase mutant according to claim 1, having no more than three of the mutations.

3. The DNA polymerase mutant according to claim 2, having only one of the mutations.

4. The DNA polymerase mutant according to claim 2, having only two of the mutations.

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

6. The DNA polymerase mutant according to claim 5, wherein the mutation at position E189 is E189K and the one or more mutations are selected from E230K, H28R, L30R, F73V, H75R, E76A, E76G, E76K, E90K, K206R, A439T, D452N, G504S, D551N, 1553V, H676R, E734G, and F749V.

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

11. The DNA polymerase mutant according to claim 10, wherein the mutation at position E189 is E189K and the one or more mutations are selected from E230K, E507K, H75R, E315K, A348V, L351F, D578N, and L552R.

12. The 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. The DNA polymerase mutant according to claim 1, which exhibits increased resistance to a DNA polymerase inhibitor selected from the group consisting of SYBR Green I dye, blood, SDS, guanidinium salts and heparin.

14. The DNA polymerase mutant according to claim 13, wherein the mutation at position E189 is E189K and the one or more mutations are selected from E230K, E507K, H28R, L30R, G38R, H75R, E76A, E76G, E76K, E90K, E315K, A439T, D452N, G504S, E507A, D551N, L552R, I553V, D578N, H676R, Q680R, E734G, E734K, and E734K.

15. The 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. The DNA polymerase mutant according to claim 1, wherein the one or more mutations include a double mutation selected from:

H28R+E507K, H28R+Q680R, E507K+Q680R, L552R+Q680R, E230K+E507K, E189K+E507K, E315K+E507K, E230K+E315K, and E507K+L552R.

17. The DNA polymerase mutant according to claim 1, wherein the mutation at position E189 is E189K and the one or more mutations are E507K and E230K.

18. 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.

19. A polymerase chain reaction method comprising contacting a target DNA with a DNA polymerase mutant according to claim 1 and one or more reagents for a DNA synthesis reaction under conditions to synthesize a new DNA complementary to the target DNA.

20. A DNA polymerase mutant obtained by a process comprising:

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

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

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

(4) repeating steps 1 to 3 until a final DNA polymerase mutant of claim 1 is obtained.

21. A process for the production of a DNA polymerase mutant according to claim 1, which process comprises:

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

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

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

(4) repeating steps 1 to 3 until a final DNA polymerase mutant of claim 1 is obtained.

22. A DNA polymerase mutant of SEQ ID NO:

13 comprising a mutation at position E189 and optionally one or more of the following mutations;

E230K, E76G, E76K, E76A, E90K, D551N, K206R, Q680R, L552R, H28R, E315K, L30R, L30P, D452N, G504S, G504D, G38R, F73V, H75R, A348V, L351F, A439T, L552R, I553V, D578N, H676R, D732G, E734G, E734K, F749V, E507K, E507A;

wherein the DNA polymerase mutant exhibits relative to wild-type DNA polymerase of SEQ ID NO;

13, and under the same conditions, increased polymerase speed, increased affinity to DNA substrate and/or increased resistance to a DNA polymerase inhibitor.

23. A DNA polymerase mutant comprising SEQ ID NO:

13 comprising a mutation at position E189 and optionally one or more of the following mutations;

E230K, E76G, E76K, E76A, E90K, D551N, K206R, Q680R, L552R, H28R, E315K, L30R, L30P, D452N, G504S, G504D, G38R, F73V, H75R, A348V, L351F, A439T, L552R, I553V, D578N, H676R, D732G, E734G, E734K, F749V, E507K, E507A;

wherein the DNA polymerase mutant exhibits relative to wild-type DNA polymerase of SEQ ID NO;

13, and under the same conditions, increased polymerase speed, increased affinity to DNA substrate and/or increased resistance to a DNA polymerase inhibitor, and wherein the DNA polymerase mutant has at least 2 and no more than 12 mutations of SEQ ID NO;

13.