cDNA synthesis improvements
View Patent ↗The present invention generally relates to methods of making cDNA molecules and cDNA libraries. The invention also relates to cDNA molecules and cDNA libraries produced according to these methods, as well as to vectors and host cells containing such cDNA molecules and libraries. The invention also relates to kits for making the cDNA molecules and libraries of the invention.
1. A method for synthesizing one or more cDNA molecules comprising combining one or more mRNA templates, or one or more poly A RNA templates and a primer with at least one polypeptide having reverse transcriptase activity and an antibody or antibody fragment inhibitor of the polypeptide having reverse transcriptase activity, incubating said template, primer, polypeptide and inhibitor at a temperature between 10° C. and 90° C., wherein said inhibitor inhibits said reverse transcriptase activity at said temperature; and elevating the temperature of said template, primer, polypeptide and inhibitor thereby inactivating said inhibitor, whereby one or more cDNA molecules are synthesized.
2. The method of claim 1 , wherein said antibody or antibody fragment is polyclonal or monoclonal.
3. The method of claim 1 , wherein said polypeptide is a reverse transcriptase selected from the group consisting of Moloney Murine Leukemia Virus reverse transcriptase (M-MLV RT), Rous Sarcoma Virus reverse transcriptase (RSV RT), Avian myeloblastosis Virus reverse transcriptase (AMV RT), Rous associated Virus reverse transcriptase (RAV RT), Myeloblastosis associated virus reverse transcriptase (MAV RT) and Human Immunodeficiency Virus reverse transcriptase (HIV RT), and fragments thereof having reverse transcriptase activity.
4. The method of claim 3 , wherein said reverse transcriptase is reduced in RNase H activity.
5. The method of claim 4 , wherein said RNase H activity is reduced to less than about 30% of RNase H activity of a corresponding wildtype reverse transcriptase.
6. The method of claim 1 , wherein said inhibitor inhibits, prevents, or reduces internal priming.
7. The method of claim 6 , wherein said temperature is within the range of about 10-65° C.
8. The method of claim 6 , wherein said temperature is within the range of about 10-55° C.
9. The method of claim 6 , wherein said temperature is within the range of about 10-45° C.
10. The method of claim 6 , wherein said temperature is within the range of about 10-35° C.
11. The method of claim 1 , wherein the primer to template ratio is between 12:1 and 1:12.
12. The method of claim 11 , wherein said primer to template ratio is between 10:1 and 1:10.
13. The method of claim 11 , wherein said primer to template ratio is between 5:1 and 1:5.
14. The method of claim 1 , wherein said primer has a length of between 20 and 100 bases.
15. The method of claim 14 , wherein said length is between 20 and 75 bases.
16. The method of claim 14 , wherein said length is between 20 and 50 bases.
17. The method of claim 14 , wherein said length is between 25 and 35 bases.
18. The method of claim 1 , wherein said polypeptide is a retroviral reverse transcriptase.
19. The method of claim 1 , wherein said polypeptide is a reverse transcriptase selected from the group consisting of M-MLV RT, RSV RT and AMV RT.
20. The method of claim 19 , wherein said reverse transcriptase is a M-MLV RT having and RNase H activity less than about 30% of the RNase H activity of the corresponding wildtype M-MLV RT.
21. Then method of claim 19 , wherein said reverse transcriptase is selected from the group consisting of SUPERSCRIPT™ (mutant M-MLV RT having reduced RNase H activity), SUPERSCRIPT™ II (mutant M-MLV RT having reduced RNase H activity), THERMOSCRIPT™ (mutant AMV RT having reduced RNase H activity) and THERMOSCRIPT™ II (mutant AMV RT having reduced RNase H activity).
22. The method of claim 1 , wherein said one or more mRNA templates is a population of mRNA templates suitable for the production of a cDNA library.
23. The method of claim 1 , wherein said cDNA molecules are a cDNA library.
24. The method of claim 1 , wherein said primer is an oligo(dT) primer.