Mutant Reverse Transcriptase and Methods of Use

Abstract

The invention relates to the generation and characterization of stable MMLV reverse transcriptase mutants. The invention also discloses methods of using stable MMLV reverse transcriptase mutants.

1 - 41 . (canceled) 
     
     
         42 . An isolated mutant MMLV reverse transcriptase having reverse transcriptase activity comprising the sequence of SEQ ID NO:19 with the exception of mutations at at least three of the following amino acid positions: E69, E302, W313, L435, N454, and M651. 
     
     
         43 . The isolated mutant MMLV reverse transcriptase of  claim 42 , wherein the reverse transcriptase comprises at least one of the following mutations:
 a glutamic acid to lysine mutation at position E69,   a glutamic acid to lysine mutation at position E302,   a glutamic acid to arginine mutation at position E302,   a tryptophan to phenylalanine mutation at position W313,   a leucine to glycine mutation at position L435,   a leucine to methionine mutation at position L435,   an asparagine to lysine mutation at position N454,   an asparagine to arginine mutation at position N454, and   a methionine to leucine mutation at position M651.   
     
     
         44 . The isolated mutant MMLV reverse transcriptase of  claim 42 , wherein the reverse transcriptase comprises a combination of mutations selected from the following groups of combinations of mutations:
 E302R/E69K/W313F/L435G/N454K;   E302R/W313F/L435G/N454K;   E302R/W313F/L435G;   E302R/E69K/N454K;   E302R/W313F; and   E69K/E302R/W313F/L435G/N454K/D524N.   
     
     
         45 . The isolated mutant MMLV reverse transcriptase of  claim 42 , further comprising a C-terminal extension. 
     
     
         46 . The isolated mutant MMLV reverse transcriptase of  claim 45 , wherein said C-terminal extension is RDRNKNNDRRKAKENE (SEQ ID NO:1). 
     
     
         47 . The isolated mutant MMLV reverse transcriptase of  claim 42 , wherein said reverse transcriptase lacks RNase H activity. 
     
     
         48 . The isolated mutant MMLV reverse transcriptase of  claim 42 , wherein the reverse transcriptase has at least one of the following characteristics: increased stability, increased accuracy, increased processivity, and increased specificity. 
     
     
         49 . A composition comprising the isolated mutant MMLV reverse transcriptase of  claim 42 . 
     
     
         50 . The composition of  claim 49 , wherein the reverse transcriptase comprises at least one of the following mutations:
 a glutamic acid to lysine mutation at position E69,   a glutamic acid to lysine mutation at position E302,   a glutamic acid to arginine mutation at position E302,   a tryptophan to phenylalanine mutation at position W313,   a leucine to glycine mutation at position L435,   a leucine to methionine mutation at position L435,   an asparagine to lysine mutation at position N454,   an asparagine to arginine mutation at position N454, and   a methionine to leucine mutation at position M651.   
     
     
         51 . The composition of  claim 49 , wherein the reverse transcriptase comprises a combination of mutations selected from the following groups of combinations of mutations:
 E302R/E69K/W313F/L435G/N454K;   E302R/W313F/L435G/N454K;   E302R/W313F/L435G;   E302R/E69K/N454K;   E302R/W313F; and   E69K/E302R/W313F/L435G/N454K/D524N.   
     
     
         52 . The composition of  claim 49 , wherein the reverse transcriptase further comprises a C-terminal extension. 
     
     
         53 . The composition of  claim 52 , wherein said C-terminal extension is RDRNKNNDRRKAKENE (SEQ ID NO:1). 
     
     
         54 . The composition of claim of  claim 49 , wherein the reverse transcriptase has at least one of the following characteristics: increased stability, increased accuracy, increased processivity, and increased specificity. 
     
     
         55 . The composition of  claim 49 , wherein said reverse transcriptase lacks RNase H activity. 
     
     
         56 . The composition of  claim 49 , further comprising an epsilon subunit from a eubacteria. 
     
     
         57 . The composition of  claim 56 , wherein said epsilon subunit is from  Escherichia coli.    
     
     
         58 . The composition of  claim 56 , wherein said epsilon subunit is epsilon 186 from  Escherichia coli.    
     
     
         59 . The composition of  claim 49 , further comprising formamide, betaine, or DMSO. 
     
     
         60 . A kit comprising the isolated mutant MMLV reverse transcriptase of  claim 42  and packaging materials thereof. 
     
     
         61 . The kit of  claim 60 , wherein the transcriptase comprises at least one of the following mutations:
 a glutamic acid to lysine mutation at position E69,   a glutamic acid to lysine mutation at position E302,   a glutamic acid to arginine mutation at position E302,   a tryptophan to phenylalanine mutation at position W313,   a leucine to glycine mutation at position L435,   a leucine to methionine mutation at position L435,   an asparagine to lysine mutation at position N454,   an asparagine to arginine mutation at position N454, and   a methionine to leucine mutation at position M651.   
     
     
         62 . The kit of  claim 60 , wherein the reverse transcriptase comprises a combination of mutations selected from the following groups of combinations of mutations:
 E302R/E69K/W313F/L435G/N454K;   E302R/W313F/L435G/N454K;   E302R/W313F/L435G;   E302R/E69K/N454K;   E302R/W313F; and   E69K/E302R/W313F/L435G/N454K/D524N.   
     
     
         63 . The kit of  claim 60 , wherein said reverse transcriptase lacks RNase H activity. 
     
     
         64 . The kit of  claim 60 , wherein said mutant MMLV reverse transcriptase further comprises a C-terminal extension. 
     
     
         65 . The kit of  claim 64 , wherein said C-terminal extension is RDRNKNNDRRKAKENE (SEQ ID NO:1). 
     
     
         66 . The kit of  claim 60 , wherein said reverse transcriptase has at least one of the following characteristics: increased stability, increased accuracy, increased processivity, and increased specificity. 
     
     
         67 . The kit of  claim 60 , further comprising an epsilon subunit from a eubacteria. 
     
     
         68 . The kit of  claim 67 , wherein said epsilon subunit is from  Escherichia coli.    
     
     
         69 . The kit of  claim 67 , wherein said epsilon subunit is epsilon 186 from  Escherichia coli.    
     
     
         70 . The kit of  claim 60 , further comprising formamide, betaine, or DMSO.