Polymerase compositions and uses
A composition having nucleic acid polymerase activity, which comprises an active nucleic acid polymerase and an excess amount of a non-functional mutant nucleic acid polymerase protein, wherein the non-functional mutant nucleic acid polymerase protein stabilizes the active nucleic acid polymerase against loss of polymerase activity.
1. A method of stabilizing an active nucleic acid polymerase against loss of polymerase activity, comprising:
contacting the active nucleic acid polymerase with an amount of a mutant nucleic acid polymerase so as to stabilize the active nucleic acid polymerase, wherein:
the amount of the mutant nucleic acid polymerase is greater than the amount of active nucleic acid polymerase;
the active nucleic acid polymerase comprises a family A or family B thermophilic polymerase catalytic domain;
the mutant nucleic acid polymerase comprises a family A or family B polymerase catalytic domain and has reduced nucleic acid synthesis activity, reduced substrate binding activity, similar thermostability, and similar enzyme surface hydrophobicity compared to its corresponding wild type nucleic acid polymerase.
2. A The method according to claim 1 , wherein the active nucleic acid polymerase is in a composition free of detergent.
3. A method of stabilizing an active nucleic acid polymerase against loss of polymerase activity, which comprises contacting the active nucleic acid polymerase with an amount of a mutant nucleic acid polymerase so as to stabilize the active nucleic acid polymerase, wherein the mutant nucleic acid polymerase protein is a mutant of the active nucleic acid polymerase, wherein the amount of the mutant nucleic acid polymerase is greater than the amount of active nucleic acid polymerase;
the active nucleic acid polymerase comprises a family A or family B thermophilic polymerase catalytic domain;
the mutant nucleic acid polymerase has reduced nucleic acid synthesis activity, reduced substrate binding activity, similar thermostability, and similar enzyme surface hydrophobicity compared to the active nucleic acid polymerase.
4. A The method according to claim 1 , wherein the nucleic acid is DNA.
5. A method of stabilizing an active nucleic acid polymerase against loss of polymerase activity, which comprises contacting the active nucleic acid polymerase with an amount of a mutant nucleic acid polymerase so as to stabilize the active nucleic acid polymerase, wherein the active nucleic acid polymerase is a thermophilic nucleic acid polymerase, wherein both the active nucleic acid polymerase and the mutant nucleic acid polymerase are thermostable, and wherein:
the amount of the mutant nucleic acid polymerase is greater than the amount of active nucleic acid polymerase;
the active nucleic acid polymerase comprises a family A or family B polymerase catalytic domain;
the mutant nucleic acid polymerase comprises a family A or family B polymerase catalytic domain and has reduced nucleic acid synthesis activity, reduced substrate binding activity, similar thermostability, and similar enzyme surface hydrophobicity compared to its corresponding wild type nucleic acid polymerase.
6. The method according to claim 1 , wherein the active nucleic acid polymerase is a bacterial or archeal nucleic acid polymerase.
7. The method according to claim 1 , wherein the thermophilic polymerase is a Taq DNA polymerase or a Thermococcus sp. DNA polymerase.
8. A The method according to claim 1 , wherein the mutant nucleic acid polymerase does not bind to the substrate for the active nucleic acid polymerase.
9. The method according to claim 1 , wherein the active nucleic acid polymerase is stabilized in a storage composition, and/or the active nucleic acid polymerase is thermostable.
10. The method according to claim 1 , wherein the active nucleic acid polymerase is stabilized in a reaction mixture for use in a reaction involving nucleic acid polymerase activity.
11. The method according to claim 1 , wherein the active nucleic acid polymerase is stabilized against an enzyme inhibitor.
12. The method of claim 1 , further comprising preparing a kit comprising the active nucleic acid polymerase and the mutant nucleic acid polymerase.
13. The method of claim 3 , further comprising preparing a kit comprising the active nucleic acid polymerase and the mutant nucleic acid polymerase.
14. The method of claim 5 , further comprising preparing a kit comprising the active nucleic acid polymerase and the mutant nucleic acid polymerase.
15. The method of claim 3 , wherein the mutant nucleic acid polymerase does not bind to the substrate of the active nucleic acid polymerase.
16. The method of claim 5 , wherein the mutant nucleic acid polymerase does not bind to the substrate of the active nucleic acid polymerase.
17. The method of claim 3 , wherein the active nucleic acid polymerase is stabilized in a storage composition, and/or the active nucleic acid polymerase is thermostable.
18. The method of claim 5 , wherein the active nucleic acid polymerase is stabilized in a storage composition.
19. The method according to claim 5 , wherein the active nucleic acid polymerase is a bacterial or archeal nucleic acid polymerase.
20. The method according to claim 5 , wherein the thermophilic nucleic acid polymerase is a Taq DNA polymerase or a Thermococcus sp. DNA polymerase.