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 variant of Bst ( Bacillus stearothermophilus ) polymerase, wherein the variant, that excludes a large fragment Bst polymerase having a wild type sequence with a 289 amino acid truncation deleted from the N-terminal end, is characterized by one or more improved properties for isothermal amplification compared with a wild type Bst polymerase or the large fragment Bst polymerase, selected from the group consisting of:
(a) an increased reaction speed at 65° C., where the increase is at least 10%
(b) an increased temperature stability in the range of 50° C.-100° C.;
(c) an increased salt tolerance in the range of 10 mM-1 M monovalent salt;
(d) an increase in storage stability at 25° C., retaining at least 50% activity over 45 weeks;
(e) an enhanced dUTP tolerance of the range of an increase of 50% to 100% dUTP; or
(f) an increased reverse transcriptase activity by at least 2 fold
wherein the variant polymerase lacks 3′ to 5′ exonuclease activity and comprises an amino acid sequence that has at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 43, 47, 48, 65, 75, 85, 86, 111, 112 and 117.
2. A variant according to claim 1 , further comprising DNA polymerase activity.
3. A variant according to claim 1 , wherein the protein is capable of replicating DNA.
4. A variant according to claim 3 , wherein the protein is capable of replicating DNA in an isothermal amplification reaction.
5. A variant according to claim 1 in a storage buffer, or a reaction buffer.
6. A variant according to claim 5 , wherein the buffer further comprises temperature dependent inhibitor of polymerase activity.
7. A variant according to claim 1 , fused to a peptide either directly or by means of a linker.
8. A variant according to claim 5 , wherein the buffer further comprises dNTPs.
9. A variant according to claim 1 , having at least two or three or four or five or six of the improved properties.
10. A DNA encoding the protein of claim 1 .
11. A host cell comprising the DNA according to claim 10 .
12. 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.
13. A method according to claim 12 , 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.
14. 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.
15. 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.
16. A method according to claim 15 , wherein the amplification reaction results in a quantitative measure of the amount of target DNA in the preparation.