Patent
The present invention relates to reverse transcription of RNA, and in particular to reverse transcription by thermostable DNA polymerases. Thermoactinomyces vulgaris and Bacillus stearothermophilus possess reverse transcriptase activity in the presence of magnesium or manganese ions. Methods, compositions, and kits for reverse transcription and RT-PCR are also provided.
1. A reaction mixture consisting of: an isolated and purified Bacillus stearothermophilus DNA polymerase, a purified RNA, and a buffer comprising magnesium ions, wherein said buffer is substantially free of manganese ions.
2. The reaction mixture of claim 1 , wherein the concentration of said magnesium ions is from about 0.1 mM to 10.0 mM.
3. The reaction mixture of claim 1 , said mixture having a pH of about 6 to 10.
4. The reaction mixture of claim 1 , wherein said Bacillus stearothermophilus DNA polymerase comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 21.
5. The reaction mixture of claim 1 , wherein said Bacillus stearothermophilus DNA polymerase comprises an amino acid sequence selected from the group consisting of SEQ ID NO:20 and SEQ ID NO: 22.
6. A kit for reverse transcription comprising:
a) a Bacillus stearothermophilus polymerase in a vessel, with a buffer comprising magnesium ions, wherein said buffer is substantially free of manganese ions.
b) purified RNA in a vessel.
7. The kit of claim 6 , further comprising instructions for reverse transcription.
8. The kit of claim 6 , wherein said buffer further comprises a surfactant.
9. The kit of claim 6 , wherein said buffer has a pH of about 6 to 10.
10. The kit of claim 6 , wherein said Bacillus stearothermophilus DNA polymerase comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 21.
11. The kit of claim 6 , wherein said Bacillus stearothermophilus DNA polymerase comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 22.
12. The kit of claim 6 , further comprising at least one additional thermostable DNA polymerase.
13. A composition comprising: an isolated and purified Bacillus stearothermophilus DNA polymerase, a purified RNA, a buffer comprising magnesium ions, wherein said buffer is substantially free of manganese ions, and wherein said composition does not include an enzyme with reverse transcriptase activity other than said Bacillus stearothermophilus DNA polymerase.
14. The composition of claim 13 , further comprising a buffering agent.
15. The composition of claim 13 , further comprising a surfactant.
16. The composition of claim 13 , further comprising at least one additional thermostable DNA polymerase.
17. A reaction mixture consisting of a reverse transcription component and an accessory factor component, wherein said reverse transcription component consists of a Bacillus stearothermophilus DNA polymerase, wherein said accessory factor component comprises dNTP and magnesium ions, and wherein said accessory factor component is substantially free of manganese ions.
18. The reaction mixture of claim 17 , wherein said accessory factor component further comprises purified RNA.
19. The reaction mixture of claim 17 , wherein said accessory factor component further comprises a buffering agent.
20. The reaction mixture of claim 17 , wherein said accessory factor component further comprises a surfactant.
21. The reaction mixture of claim 17 , further comprising at least one additional thermostable DNA polymerase.
22. A reaction mixture comprising a Bacillus stearothermophilus DNA polymerase polymerizing a reverse transcription reaction of an RNA substrate in the presence of a buffer that is substantially free of manganese ions.
23. The reaction mixture of claim 22 , further comprising magnesium ions, and wherein the rate of said reverse transcription reaction by said Bacillus stearothermophilus DNA polymerase is greater than 50-fold higher than possible with native Taq DNA polymerase, Tth DNA polymerase, and Tne DNA polymerase in said reaction mixture.