Variant DNA polymerases having improved properties and method for improved isothermal amplification of a target DNA
Variants of the bacteriophage B103 DNA polymerase are described herein. The variant has improved properties, that include when compared to wild-type Phi29 DNA polymerase, at least one of the following: increased thermostability, improved reaction rate for DNA amplification, reduced background and a reduction of bias. Methods of using the DNA polymerase variant are also described herein.
1. A DNA polymerase comprising an amino acid sequence that has at least 95% sequence identity with SEQ ID NO:2 and at least one amino acid substitution selected from the group consisting of: A147K, R221Y, A318G, A503M, I511V, R544K and T550K.
2. The DNA polymerase according to claim 1 , further comprising at least 2 amino acid substitutions selected from the group consisting of: A147K, R221Y, A318G, A503M, I511V, R544K and T550K.
3. The DNA polymerase according to claim 1 , further comprising at least 3 amino acid substitutions selected from the group consisting of: A147K, R221Y, A318G, A503M, I511V, R544K and T550K.
4. The DNA polymerase according to claim 1 , further comprising at least 4 amino acid substitutions selected from the group consisting of: A147K, R221Y, A318G, A503M, I511V, R544K and T550K.
5. The DNA polymerase according to claim 1 , further comprising at least 5 amino acid substitutions selected from the group consisting of: A147K, R221Y, A318G, A503M, I511V, R544K and T550K.
6. The DNA polymerase according to claim 1 , further comprising at least 6 amino acid substitutions selected from the group consisting of: A147K, R221Y, A318G, A503M, I511V, R544K and T550K.
7. The DNA polymerase according to claim 1 , further comprising 7 amino acid substitution that consist of A147K, R221Y, A318G, A503M, I511V, R544K and T550K.
8. The DNA polymerase according to claim 1 , further comprising substitutions at one or more positions selected from 339, 359, 383 and 384.
9. The DNA polymerase according to claim 1 , wherein the DNA polymerase is a fusion protein.
10. A DNA polymerase composition comprising at least 99% sequence identity with SEQ ID NO:5.
11. A kit comprising:
(i) the DNA polymerase according to claim 1 ; and
(ii) a reaction buffer.
12. The kit of claim 11 , wherein the DNA polymerase is lyophilized and/or the reaction buffer is in concentrated form.
13. The kit of claim 11 , further comprising one or more primers.
14. The kit of claim 13 , whereon the primers are selected from the group consisting of: random primers, exonuclease-resistant primers and primers having chemical modifications.
15. The kit of claim 1 , further comprising one or more dNTPs, crowding agents and/or cationic, anionic, zwitterionic or non-ionic detergents.
16. A method for improved isothermal amplification of a target DNA, comprising:
preparing a reaction mix comprising the target DNA with a DNA polymerase of claim 1 , dNTPs and, optionally, one or more primers; and amplifying the target DNA isothermally.
17. The method according to claim 16 , further comprising amplifying the target DNA at an amplification temperature in the range of 30° C.-42° C.
18. The method according to claim 16 , wherein the target DNA is linear or circular.
19. The method according to claim 16 , wherein the target DNA is a whole genome or a target DNA sequence therein.
20. The method according to claim 16 , further comprising amplifying the target DNA at a temperature in the range of 30° C.-42° C.; and obtaining a higher yield within 2 hours of amplified DNA compared to the yield obtained with wild-type Phi29 DNA polymerase under the same reaction conditions.
21. The method according to claim 16 , further comprising amplifying the target DNA at a temperature in the range of 30° C.-42° C. and obtaining a higher rate of amplification compared to the rate of amplification using wild-type Phi29 DNA polymerase under the same reaction conditions.
22. The method according to claim 16 , further comprising amplifying the target DNA at a temperature in the range of 30° C.-42° C. and obtaining a more even representation of the substrate DNA compared to the biased representation of wild-type Phi29 DNA polymerase under the same reaction conditions.
23. The method according to claim 16 , further comprising amplifying the target DNA at a temperature in the range of 30° C.-42° C., and obtaining reduced background compared to amplifying the substrate DNA with wild-type Phi29 DNA polymerase under the same reaction conditions.
24. The method according to claim 16 , further comprising amplifying the target DNA in a high salt buffer.