Methods of using stabilized compositions of thermostable DNA polymerase and anionic detergent
The present invention provides compositions, methods, and kits for protecting thermostable DNA polymerase during amplification reactions conducted at a temperature ranging from about 40° C. to greater than 100° C. The composition comprises a thermostable DNA polymerase and an anionic detergent or zwitterionic detergent.
1. A method for inhibiting inactivation of a thermostable DNA polymerase in a thermal cycling process, the method comprising contacting the thermostable DNA polymerase with an anionic detergent chosen from poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether potassium salt, poly(ethylene glycol) monolaurate, polyoxyethylene(150)dinonylphenyl ether, and nonyl nonoxynol-15 phosphate during the thermal cycling process.
2. The method of claim 1 , wherein the anionic detergent is poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether potassium salt.
3. The method of claim 1 , wherein the thermostable DNA polymerase and the anionic detergent are formulated as a mixture comprising from about 0.001% to about 1% by weight of the anionic detergent.
4. The method of claim 1 , wherein the thermostable DNA polymerase is from a thermophilic bacterium chosen from Thermus flavus, Thermus ruber, Thermus thermophilus, Bacillus stearothermophilus, Thermus aquaticus, Thermus lacteus, Thermus rubens , and Methanothermus fervidus .
5. The method of claim 1 , wherein the thermostable DNA polymerase is Taq.
6. The method of claim 1 , wherein the thermal cycling process is conducted at a temperature ranging from about 40° C. to about 100° C. for approximately 2 to 50 cycles.
7. The method of claim 1 , further comprising a buffering agent such that the thermal cycle process is maintained at a pH from about 6 to about 9, the buffering agent being chosen from MOPS, HEPES, TAPS, Bicine, Tricine, TES, PIPES, MES, and Tris.
8. The method of claim 1 , further comprising a salt chosen from potassium chloride, sodium chloride, magnesium chloride, calcium chloride, lithium chloride, and combinations thereof.
9. The method of claim 1 , further comprising a mixture of dNTPs chosen from dATP, dCTP, dGTP, and dTTP.
10. The method of claim 1 , further comprising an agent that increases the efficiency of PCR, the agent being chosen from dimethyl sulfoxide, formamide, and betaine.
11. The method of claim 1 , further comprising at least one target polynucleotide and at least one pair of oligonucleotide primers.
12. The method of claim 1 , wherein the thermal cycle process has an efficiency of amplification of at least about 50%.
13. The method of claim 1 , wherein the thermostable DNA polymerase is Taq and the anionic detergent is poly(ethylene glycol) 4-nonylphenyl 3-sulfopropyl ether potassium salt.
14. The method of claim 13 , wherein the thermostable DNA polymerase and the anionic detergent are formulated as a mixture comprising from about 0.001% to about 1% by weight of the anionic detergent.
15. The method of claim 13 , wherein the thermal cycling process is conducted at a temperature ranging from about 50° C. to about 95° C. for approximately 10 to 40 cycles.
16. The method of claim 13 , further comprising a buffering agent such that the thermal cycle process is maintained at a pH from about 7 to about 8, the buffering agent being chosen from MOPS, HEPES, TAPS, Bicine, Tricine, TES, PIPES, MES, and Tris; a salt chosen from potassium chloride, sodium chloride, magnesium chloride, calcium chloride, lithium chloride, and combinations thereof; and a mixture of dNTPs chosen from dATP, dCTP, dGTP, and dTTP.
17. The method of claim 13 , further comprising an agent that increases the efficiency of PCR, the agent being chosen from dimethyl sulfoxide, formamide, and betaine.
18. The method of claim 13 , further comprising at least one target polynucleotide and at least one pair of oligonucleotide primers.
19. The method of claim 13 , wherein the thermal cycle process has an efficiency of amplification of at least about 65%.