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 has at least 98% sequence identity with at least one sequence selected from the group consisting of SEQ ID NOs: 44, 45, 54, 55, 64, 77, 88, 90, 91, 92 and 106.
2. A protein according to claim 1 , further comprising DNA polymerase activity.
3. A protein according to claim 1 , wherein the protein is capable of replicating DNA.
4. A protein according to claim 3 , wherein the protein is capable of replicating DNA in an isothermal amplification reaction.
5. A protein according to claim 1 in a storage buffer, or a reaction buffer.
6. A protein according to claim 5 , wherein the buffer further comprises temperature dependent inhibitor of polymerase activity.
7. A protein according to claim 1 , fused to a peptide either directly or by means of a linker.
8. A protein according to claim 1 , wherein the buffer further comprises dNTPs.
9. A DNA encoding the protein of claim 1 .
10. A host cell comprising the DNA according to claim 9 .
11. 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.
12. A method according to claim 11 , 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.
13. 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.
14. 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.
15. A method according to claim 14 , wherein the amplification reaction results in a quantitative measure of the amount of target DNA in the preparation.
16. 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.
17. A protein according to claim 16 , having at least two or three or four or five or six of the improved properties.