Recombinant reverse transcriptases
View Patent ↗The present invention relates to a gene that encodes a hyperactive reverse transcriptase having DNA polymerase activity and substantially reduced RNase H activity, vectors containing the gene and host cells transformed with the invention. The present invention also includes a method of producing the hyperactive reverse transcriptase, producing cDNA from mRNA using the reverse transcriptase of the invention, kits and assay templates made using the hyperactive reverse transcriptase.
1. An isolated reverse transcriptase protein comprising SEQ ID NO:2.
2. The reverse transcriptase of claim 1 , wherein the reverse transcriptase comprises reverse transcriptase produced recombinantly.
3. The reverse transcriptase of claim 1 , wherein the reverse transcriptase is purified and is greater than 90% pure.
4. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a yield of greater than about 1 ug of an amplified RNA (aRNA) from 100 ng of template RNA in a single amplification reaction.
5. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a yield of greater than about 5 ug of an aRNA from 100 ng of template RNA in a single amplification reaction.
6. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a yield of greater than about 7 ug of an aRNA from 100 ng of template RNA in a single amplification reaction.
7. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a yield of greater than about 10 ug of an aRNA from 100 ng of template RNA in a single amplification reaction.
8. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a yield of greater than about 15 ug of an aRNA from 100 ng of template RNA in a single amplification reaction.
9. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a yield of greater than about 25 ug of an aRNA from 100 ng of template RNA in a single amplification reaction.
10. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a yield of greater than about 1 ug of an aRNA from 10 pg of template RNA after a two-round amplification reaction.
11. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a yield of greater than about 2 ug of an aRNA from 10 pg of template RNA after a two-round amplification reaction.
12. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a yield of greater than about 5 ug of an aRNA from 10 pg of template RNA after a two-round amplification reaction.
13. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a yield of greater than about 10 ug of an aRNA from 10 pg of template RNA after a two-round amplification reaction.
14. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a cDNA greater than about 6, 9 or 11 kilobases in a single cDNA synthesis reaction.
15. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a cDNA greater than about 6 to about 15 kilobases in a single cDNA synthesis reaction.
16. The reverse transcriptase of claim 1 , wherein the reverse transcriptase produces a cDNA greater than about 15 kilobases in a single cDNA synthesis reaction.
17. The reverse transcriptase of claim 1 , wherein the DNA polymerase activity is greater than about 200 Units per microgram.
18. The reverse transcriptase of claim 1 , wherein the DNA polymerase activity is between about 0.1 and 300 Units per microgram.
19. The reverse transcriptase of claim 1 , wherein the RNase H activity is between about 0.1 and about 25 percent of the wild-type MMLV RNase H activity.
20. A kit for nucleic acid synthesis, comprising, in a suitable container:
a reverse transcriptase protein of claim 1 ; and
a reaction solution for the reverse transcriptase protein.
21. The kit of claim 20 , further comprising an insert that comprises information for using the reverse transcriptase protein.
22. The kit of claim 20 , wherein thereaction solution comprises a concentrated reverse transcriptase reaction buffer.
23. The kit of claim 20 , further comprising a primer.
24. The kit of claim 20 , wherein the reaction solution comprises a reverse transcriptase buffer.
25. The kit of claim 20 , wherein the reaction solution comprises a PCR buffer.
26. The kit of claim 20 , further comprising a mix of nucleotides.
27. The kit of claim 20 , further comprising containers comprising individual nucleotides.
28. The kit of claim 20 , wherein the reaction solution comprises a buffer for in vitro transcription.
29. The kit of claim 20 , further comprising a template purification column.
30. The kit of claim 20 , further comprising magnetic particles suitable for nucleic acid purification.
31. A kit for RNA amplification, comprising, in a suitable container: a reverse transcriptase protein comprising SEQ ID NO:2; an oligonucleotide comprising a transcriptional promoter region and oligo(dT) region; a DNA polymerase; and an RNA polymerase.
32. The kit of claim 31 , further comprising an insert that comprises information for using the reverse transcriptase protein.
33. The kit of claim 31 , further comprising a primer.
34. The kit of claim 31 , further comprising a reverse transcriptase buffer.
35. The kit of claim 31 , further comprising a DNA Polymerase buffer.
36. The kit of claim 31 , further comprising a mix of nucleotides.
37. The kit of claim 31 , further comprising containers comprising individual nucleotides.
38. The kit of claim 31 , further comprising a buffer for in vitro transcription.
39. The kit of claim 31 , further comprising a nucleic acid purification column.
40. The kit of claim 31 , further comprising a magnetic particle or particles suitable for nucleic acid purification.
41. An RT-PCR kit comprising in one or more suitable containers: a reverse transcriptase comprising SEQ ID NO:2, two or more primers, nucleotides, a thermostable DNA polymerase and an RT-PCT buffer.
42. The RT-PCR kit of claim 41 , wherein the container comprising a reverse transcriptase further comprises one or more further reverse transcriptases in addition to the reverse transcriptase comprising SEQ ID NO:2.