DNA polymerases
View Patent ↗Novel proteins having DNA polymerase are described which have utility in amplification reactions and have improved properties over Bst polymerase such as for example enhanced reverse transcriptase activity.
1. A protein, comprising an amino acid sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 47, 48, 64, 75, 85, 86, 111, 112 and 117 or comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 65.
2. A protein according to claim 1 , wherein the amino acid sequence is SEQ ID. No. 47.
3. A protein according to claim 1 , wherein the amino acid sequence is SEQ ID. No. 48.
4. A protein according to claim 1 , wherein the amino acid sequence is SEQ ID. No. 64.
5. A protein according to claim 1 , wherein the amino acid sequence is SEQ ID. No. 65.
6. A protein according to claim 1 , wherein the amino acid sequence is SEQ ID. No. 75.
7. A protein according to claim 1 , wherein the amino acid sequence is SEQ ID. No. 85.
8. A protein according to claim 1 , wherein the amino acid sequence is SEQ ID. No. 86.
9. A protein according to claim 1 , wherein the amino acid sequence is SEQ ID. No. 111.
10. A protein according to claim 1 , wherein the amino acid sequence is SEQ ID. No. 112.
11. A protein according to claim 1 , wherein the amino acid sequence is SEQ ID. No. 117.
12. A protein according to claim 1 , further comprising DNA polymerase activity.
13. A protein according to claim 1 , wherein the protein is capable of replicating DNA.
14. A protein according to claim 13 , wherein the protein is capable of replicating DNA in an isothermal amplification reaction.
15. A protein according to claim 1 in a storage buffer, or a reaction buffer.
16. A protein according to claim 15 , wherein the buffer further comprises temperature dependent inhibitor of polymerase activity.
17. A protein according to claim 1 , fused to a peptide either directly or by means of a linker.
18. A protein according to claim 16 , wherein the buffer further comprises dNTPs.
19. A DNA encoding the protein of claim 1 .
20. A host cell comprising the DNA according to claim 19 .
21. A method for determining whether a protein according to claim 1 has improved polymerase activity compared with a wild type Bst polymerase; comprising synthesizing a protein according to claim 1 ; and determining the polymerase activity.
22. A method according to claim 21 , wherein characterizing the polymerase activity further comprises: determining in comparison with a wild type Bst polymerase, at last one improved property selected from the group consisting of: thermostability; stability in storage; tolerance to salt; performance in isothermal amplification; strand displacement; kinetics; processivity; fidelity; altered ribonucleotide incorporation; 2′-deoxyuridine 5′-triphosphate incorporation; reverse transcriptase activity; and modified nucleotide incorporation.
23. A method, comprising:
(a) selecting a protein according to claim 1 ; and
(b) expressing the protein as a fusion protein with an additional peptide at an end of the amino acid sequence, the additional peptide attached either directly or by means of a linker.
24. A method of isothermal amplification, comprising:
(a) providing a reaction mixture comprising a protein according to claim 1 , primers and dNTPs;
(b) combining a target DNA with the preparation; and
(c) amplifying the target DNA at a temperature less than 90° C.
25. A method according to claim 24 , wherein the amplification reaction results in a quantitative measure of the amount of target DNA in the preparation.
26. A protein according to claim 1 further characterized by one or more improved properties for isothermal amplification compared with a wild type Bst polymerase, selected from the group consisting of:
(a) an increased reaction speed where the increase is at least 10% and as much as 200%, 500% or 1000%;
(b) an increased temperature stability in the range of 50° C.-100° C., 50° C.-90° C. or 60° C.-90° C.;
(c) an increased salt tolerance in the range of 10 mM-1 M, or 20 mM-200 or 500 mM monovalent salt;
(d) an increase in storage stability at 25° C., retaining at least 50% activity over 45, weeks, over 1 year or over 2 years;
(e) an enhanced dUTP tolerance of the range of an increase of 50% to 100% dUTP; and
(f) an increased reverse transcriptase activity by at least 2 fold.
27. A protein according to claim 26 , having at least two or three or four or five or six of the improved properties.