Polymerase compositions and uses thereof
View Patent ↗The subject invention provides novel compositions containing a mixture of (a) an enzyme that possesses substantial 3′–5′ exonuclease activity (b) a DNA polymerase with less 3′–5′ exonuclease activity than the enzyme with substantial 3′–5′ exonuclease activity. Preferably, the DNA polymerase for inclusion in the compositions are DNA polymerases that substantially lack 3′–5′ exonuclease activity. A preferred embodiment of the invention is a composition comprising the Taq DNA polymerase (isolated from Thermus aquaticus ) and the Pfu DNA polymerase (isolated from Pyrococcus furiosus ). Another aspect of the invention is to provide methods for synthesizing polynucleotides, typically DNA, using compositions comprising an enzyme that possesses substantial 3′–5′ exonuclease activity and a DNA polymerase with less 3′–5′ exonuclease activity than the enzymes possessing substantial 3′–5′ exonuclease activity, preferably a DNA polymerase that substantially lacks 3′–5′ exonuclease activity. Another aspect of the invention involves the use the subject method of polynucleotide synthesis to carry out the synthesis step in a polymerase chain reaction experiment. Yet another aspect of the invention is to provide kits for the synthesis of polynucleotides, wherein the kits comprise an enzyme that possesses substantial 3′–5′ exonuclease activity and a DNA polymerase with less 3′–5′ exonuclease activity than the enzyme possessing substantial 3′–5′ exonuclease activity.
1. A kit for the synthesis of a polynucleotide, said kit comprising:
a composition comprising:
(a) a first DNA polymerase, wherein said first polymerase possesses 3′–5′ exonuclease activity and is thermostable, and
(b) a second DNA polymerase, wherein said second polymerase substantially lacks 3′–5′ exonuclease activity and is thermostable;
wherein both the first DNA polymerase and the second DNA polymerase retain at least 50 percent of their specific activity after exposure to a temperature of 80 degrees Celsius for a period of 20 minutes.
2. A method of amplifying a polynucleotide sequence, said method comprising mixing a composition with a synthesis primer and a synthesis template, said composition comprising
(a) a first DNA polymerase possessing 3′–5′ exonuclease activity, wherein said first polymerase is thermostable, and
(b) a second DNA polymerase, wherein said second polymerase substantially lacks 3′–5′ exonuclease activity and is thermostable;
wherein both the first DNA polymerase and the second DNA polymerase retain at least 50 percent of their specific activity after exposure to a temperature of 80 degrees Celsius for a period of 20 minutes.
3. A method according to claim 2 , wherein said first DNA polymerase is selected from the group consisting of Pyrococcus furiosus DNA polymerase, Thermotoga maritima DNA polymerase, Thermococcus litoralis DNA polymerase, and Pyrococcus GB-D DNA polymerase.
4. A method according to claim 3 , wherein said first DNA polymerase is Pyrococcus furiosus DNA polymerase.
5. A method according to claim 2 , wherein the second DNA polymerase is selected from the group consisting of Thermus aquaticus DNA polymerase, (exo−) Thermococcus litoralis DNA polymerase, (exo−) Pyrococcus furiosus DNA polymerase, and (exo−) Pyrococcus GB-D DNA polymerase.
6. A method according to claim 2 , wherein said second DNA polymerase is Thermus aquaticus DNA polymerase.
7. A method according to claim 4 , wherein said second DNA polymerase is Thermus aquaticus DNA polymerase.
8. A kit according to claim 1 , wherein said first DNA polymerase is selected from the group consisting of Pyrococcus furiosus DNA polymerase, Thermotoga maritima DNA polymerase, Thermococcus litoralis DNA polymerase, and Pyrococcus GB-D DNA polymerase.
9. A kit according to claim 8 , wherein said first DNA polymerase is Pyrococcus furiosus DNA polymerase.
10. A kit according to claim 1 , wherein the second DNA polymerase is selected from the group consisting of Thermus aquaticus DNA polymerase, (exo−) Thermococcus litoralis DNA polymerase, (exo−) Pyrococcus furiosus DNA polymerase, and (exo−) Pyrococcus GB-D DNA polymerase.
11. A kit according to claim 10 , wherein said second DNA polymerase is Thermus aquaticus DNA polymerase.
12. A kit according to claim 1 , said kit further comprising DNA primers.
13. A composition comprising:
(a) a first DNA polymerase, wherein said first polymerase possesses 3′–5′ exonuclease activity and is thermostable, and
(b) a second DNA polymerase, wherein said second polymerase substantially lacks 3′–5′ exonuclease activity and is thermostable;
wherein both the first DNA polymerase and the second DNA polymerase retain at least 50 percent of their specific activity after exposure to a temperature of 80 degrees Celsius for a period of 20 minutes.
14. A composition according to claim 13 , wherein said second DNA polymerase is Thermus aquaticus DNA polymerase.
15. A composition according to claim 13 , wherein said first DNA polymerase is selected from the group consisting of Pyrococcus furiosus DNA polymerase, Thermotoga maritima DNA polymerase, Thermococcus litoralis DNA polymerase, and Pyrococcus GB-D DNA polymerase.
16. A composition according to claim 14 , wherein said first DNA polymerase is Pyrococcus furiosus DNA polymerase.
17. A composition according to claim 15 , wherein said first DNA polymerase is Thermococcus litoralis DNA polymerase.
18. A composition according to claim 15 , wherein said first DNA polymerase is Pyrococcus GB-D DNA polymerase.
19. A composition according to claim 15 , wherein said first DNA polymerase is Thermotoga maritima DNA polymerase.
20. A composition according to claim 17 , wherein the second DNA polymerase is Thermus aquaticus DNA polymerase.
21. A composition according to claim 17 , wherein the second DNA polymerase is (exo−) Thermococcus litoralis DNA polymerase.
22. A composition according to claim 17 , wherein the second DNA polymerase is (exo−) Pyrococcus furiosus DNA polymerase.
23. A composition according to claim 17 , wherein the second DNA polymerase is (exo−) Pyrococcus GB-D DNA polymerase.
24. A composition according to claim 18 , wherein the second DNA polymerase is Thermus aquaticus DNA polymerase.
25. A composition according to claim 18 , wherein the second DNA polymerase is (exo−) Thermococcus litoralis DNA polymerase.
26. A composition according to claim 18 , wherein the second DNA polymerase is (exo−) Pyrococcus furiosus DNA polymerase.
27. A composition according to claim 18 , wherein the second DNA polymerase is (exo−) Pyrococcus GB-D DNA polymerase.
28. A composition according to claim 19 , wherein the second DNA polymerase is Thermus aquaticus DNA polymerase.
29. A composition according to claim 19 , wherein the second DNA polymerase is (exo−) Thermococcus litoralis DNA polymerase.
30. A composition according to claim 19 , wherein the second DNA polymerase is (exo−) Pyrococcus furiosus DNA polymerase.
31. A composition according to claim 19 , wherein the second DNA polymerase is (exo−) Pyrococcus GB-D DNA polymerase.
32. A method of synthesizing a polynucleotide sequence, said method comprising mixing a composition with a synthesis primer and a synthesis template, said composition comprising
(a) a first DNA polymerase possessing 3′–5′ exonuclease activity, wherein said first polymerase is thermostable, and
(b) a second DNA polymerase, wherein said second polymerase substantially lacks 3′–5′ exonuclease activity and is thermostable;
wherein both the first DNA polymerase and the second DNA polymerase retain at least 50 percent of their specific activity after exposure to a temperature of 80 degrees Celsius for a period of 20 minutes.
33. A method according to claim 32 , wherein said first DNA polymerase is selected from the group consisting of Pyrococcus furiosus DNA polymerase, Thermotoga maritima DNA polymerase, Thermococcus litoralis DNA polymerase, and Pyrococcus GB-D DNA polymerase.
34. A method according to claim 33 , wherein said first DNA polymerase is Pyrococcus furiosus DNA polymerase.
35. A method according to claim 32 , wherein the second DNA polymerase is selected from the group consisting of Thermus aquaticus DNA polymerase, (exo−) Thermococcus litoralis DNA polymerase, (exo−) Pyrococcus furiosus DNA polymerase, and (exo−) Pyrococcus GB-D DNA polymerase.
36. A method according to claim 32 , wherein said second DNA polymerase is Thermus aquaticus DNA polymerase.
37. A method according to claim 33 , wherein the second DNA polymerase is selected from the group consisting of Thermus aquaticus DNA polymerase, (exo−) Thermococcus litoralis DNA polymerase, (exo−) Pyrococcus furiosus DNA polymerase, and (exo−) Pyrococcus GB-D DNA polymerase.
38. A method according to claim 34 , wherein said second DNA polymerase is Thermus aquaticus DNA polymerase.
39. A method according to claim 32 , wherein the first DNA polymerase is selected from the group consisting of Pyrococcus furiosus DNA polymerase, Thermococcus litoralis DNA polymerase, and Pyrococcus GB-D DNA polymerase.