Thermostable reverse transcriptases and uses thereof

Abstract

The present invention is in the fields of molecular and cellular biology. The invention is generally related to reverse transcriptase enzymes and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to reverse transcriptase enzymes which have been mutated or modified to increase thermostability, decrease terminal deoxynucleotidyl transferase activity, and/or increase fidelity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these reverse transcriptase enzymes or compositions. The invention also relates to nucleic acid molecules produced by these methods and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also concerns kits comprising such enzymes or compositions.

1 . A retroviral reverse transcriptase which has been modified or mutated to increase or enhance thermostability relative to the corresponding wild type reverse transciptase.  
     
     
         2 . The reverse transcriptase of  claim 1 , which retains at least 50% of reverse transcriptase activity after heating to 50° C. for 5 minutes.  
     
     
         3 . The reverse transcriptase of  claim 1 , which retains at least 70% of reverse transcriptase activity after heating to 50° C. for 5 minutes.  
     
     
         4 . The reverse transcriptase of  claim 1 , which retains at least 85% of reverse transcriptase activity after heating to 50° C. for 5 minutes.  
     
     
         5 . The reverse transcriptase of  claim 1 , which retains at least 95% of reverse transcriptase activity after heating to 50° C. for 5 minutes.  
     
     
         6 . The reverse transcriptase of  claim 1 , wherein the reverse transcriptase exhibits increased fidelity relative to the corresponding wild type reverse transcriptase.  
     
     
         7 . The reverse transcriptase of  claim 1 , wherein the reverse transcriptase exhibits reduced or substantially reduced terminal deoxynucleotidyl transferase activity relative to the corresponding wild type reverse transcriptase.  
     
     
         8 . The reverse transcriptase of  claim 1 , wherein the reverse transcriptase exhibits reduced or substantially reduced RNase H activity relative to the corresponding wild type reverse transcriptase.  
     
     
         9 . The reverse transcriptase of  claim 1 , wherein the reverse transcriptase is selected from the group consisting of M-MLV, RSV, AMV, and HIV reverse transcriptases.  
     
     
         10 . The reverse transcriptase of  claim 9 , which is M-MLV reverse transcriptase.