High fidelity reverse transcriptases and uses thereof

Abstract

The invention relates to reverse transcriptases which have increased fidelity (or reduced misincorporation rate) and/or terminal deoxynucleotidyl transferase activity. In particular, the invention relates to a method of making such reverse transcriptases by modifying or mutating specified positions in the reverse transcriptases. The invention also relates to nucleic acid molecules containing the genes encoding the reverse trancriptases of the invention, to host cells containing such nucleic acid molecules and to methods to make the reverse trancriptases using the host cells. The reverse transcriptases of the invention are particularly suited for nucleic acid synthesis, sequencing, amplification and cDNA synthesis.

1 . A reverse transcriptase which has been modified or mutated by at least one way selected from the group consisting of: 
 (a) to increase or enhance fidelity;    (b) to reduce or eliminate misincorporation of nucleotides during nucleic acid synthesis; and    (c) to decrease or eliminate terminal deoxynucleotidyl transferase activity,    wherein said reverse transcriptase is not derived from a Human Immunodeficiency Virus.    
     
     
         2 . The reverse transcriptase of  claim 1 , wherein said reverse transcriptase is modified or mutated to increase or enhance fidelity.  
     
     
         3 . The reverse transcriptase of  claim 1 , wherein said reverse transcriptase is modified or mutated to reduce or eliminate misincorporation of nucleotides during nucleic acid synthesis.  
     
     
         4 . The reverse transcriptase of  claim 1 , wherein said reverse transcriptase is modified or mutated to decrease or eliminate terminal deoxynucleotidyl transferase activity.  
     
     
         5 . The reverse transcriptase 1, wherein said reverse transcriptase is modified or mutated to increase or enhance fidelity, and to reduce or eliminate misincorporation of nucleotides during nucleic acid synthesis.  
     
     
         6 . The reverse transcriptase of  claim 1 , wherein said reverse transcriptase is modified or mutated to increase or enhance fidelity, and to decrease or eliminate terminal deoxynucleotidyl transferase activity.  
     
     
         7 . The reverse transcriptase of  claim 1 , wherein said reverse transcriptase is modified or mutated to reduce or eliminate misincorporation of nucleotides during nucleic acid synthesis, and to decrease or eliminate terminal deoxynucleotidyl transferase activity.  
     
     
         8 . The reverse transcriptase of  claim 1 , wherein said reverse transcriptase is further modified or mutated to reduce or substantially reduce RNase H activity.  
     
     
         9 . The reverse transcriptase of  claim 1 , wherein said reverse transcriptase is derived from a virus selected from the group consisting of M-MLV, RSV and AMV.  
     
     
         10 . The reverse transcriptase of  claim 8 , wherein said reverse transcriptase is derived from a reverse transcriptase selected from the group consisting of M-MLV H −  reverse transcriptase, RSV H −  reverse transcriptase, AMV H −  reverse transcriptase and RAV H −  reverse transcriptase.  
     
     
         11 . The reverse trancriptase of  claim 9 , wherein said modified or mutated M-MLV has a mutation or modification at position Tyr64.  
     
     
         12 . The reverse trancriptase of  claim 11 , wherein Tyr64 is replaced with a tryptophan.  
     
     
         13 . The reverse trancriptase of  claim 9 , wherein said modified or mutated M-MLV has a mutation or modification at position Arg116.  
     
     
         14 . The reverse trancriptase of  claim 13 , wherein Arg116 is replaced with a methionine.  
     
     
         15 . The reverse trancriptase of  claim 9 , wherein said modified or mutated M-MLV has a mutation or modification at position Lys152.  
     
     
         16 . The reverse trancriptase of  claim 15 , wherein Lys152 is replaced with an arginine.  
     
     
         17 . The reverse trancriptase of  claim 9 , wherein said modified or mutated M-MLV has a mutation or modification at position Glu190.  
     
     
         18 . The reverse trancriptase of  claim 17 , wherein Glu190 is replaced with a phenylalanine.  
     
     
         19 . The reverse trancriptase of  claim 9 , wherein said modified or mutated M-MLV has a mutation or modification at position Thr197.  
     
     
         20 . The reverse trancriptase of  claim 19 , wherein Thr197 is replaced with an alanine.