Novel reverse transcriptases for use in high temperature nucleic acid synthesis

Abstract

The invention provides novel reverse transcriptases (RTs) with desirable properties such as increased thermostability, increased thermoreactivity and/or increased resistance to inhibitors. In certain embodiments, the invention provides methods of producing, amplifying and/or sequencing nucleic acid molecules (particularly cDNA molecules) using kits, compositions and/or reactions mixtures containing such novel reverse transcriptase enzymes.

1 - 106 . (canceled) 
     
     
         107 . A mutant M-MLV reverse transcriptase, comprising at least six mutations at an amino acid position selected from the group: P51, E69, H204, F309, W313, T330, L435, N454, D524, D583, H594, D653, and L671 of wild type M-MLV (SEQ ID NO:2). 
     
     
         108 . The mutant M-MLV reverse transcriptase according to  claim 107 , wherein said mutations are selected from the group: P51L, E69K, H204R, F309N, W313F, T330P, L435G, N454K, D524G, D583N, H594Q, D653N, and L671P. 
     
     
         109 . The mutant M-MLV reverse transcriptase according to  claim 107 , further comprising at least one mutation at an amino acid position selected from the group: S67, T197 and E302. 
     
     
         110 . The mutant M-MLV reverse transcriptase according to  claim 109 , wherein said at least one mutation is selected from the group: S67R, S67N, S67K, T197A, T1975, T197G, E302K, E302R, and E302G. 
     
     
         111 . A composition for nucleic acid synthesis comprising a buffer and mutant M-MLV reverse transcriptase according to  claim 107 . 
     
     
         112 . The composition according to  claim 111 , wherein said mutations are selected from the group: P51L, E69K, H204R, F309N, W313F, T330P, L435G, N454K, D524G, D583N, H594Q, D653N, and L671P. 
     
     
         113 . The composition according to  claim 111 , further comprising at least one mutation at an amino acid position selected from the group: S67, T197 and E302. 
     
     
         114 . The composition according to  claim 113 , wherein said at least one mutation is selected from the group: S67R, S67N, S67K, T197A, T1975, T197G, E302K, E302R, and E302G. 
     
     
         115 . A kit comprising, in one or more packaged containers, a mutant M-MLV reverse transcriptase according to  claim 107 . 
     
     
         116 . The kit according to  claim 115 , wherein said mutations are selected from the group: P51L, E69K, H204R, F309N, W313F, T330P, L435G, N454K, D524G, D583N, H594Q, D653N, and L671P. 
     
     
         117 . The kit according to  claim 115 , further comprising at least one mutation at an amino acid position selected from the group: S67, T197 and E302. 
     
     
         118 . The kit according to  claim 117 , wherein said at least one mutation is selected from the group: S67R, S67N, S67K, T197A, T1975, T197G, E302K, E302R, and E302G. 
     
     
         119 . A mutant M-MLV reverse transcriptase that is at least 70% identical to SEQ ID NO: 2, wherein said mutant M-MLV reverse transcriptase comprises six or more mutations selected from the list P51, E69, H204, F309, W313, T330, L435, N454, D524, D583, H594, D653, and L671. 
     
     
         120 . The mutant M-MLV reverse transcriptase according to  claim 119 , wherein said M-MLV reverse transcriptase is at least 80% identical to SEQ ID NO: 2. 
     
     
         121 . The mutant M-MLV reverse transcriptase according to  claim 119 , wherein said M-MLV reverse transcriptase is at least 90% identical to SEQ ID NO: 2. 
     
     
         122 . The mutant M-MLV reverse transcriptase according to  claim 119 , wherein said mutations are selected from the group: P51L, E69K, H204R, F309N, W313F, T330P, L435G, N454K, D524G, D583N, H594Q, D653N, and L671P. 
     
     
         123 . The mutant M-MLV reverse transcriptase according to  claim 119 , further comprising at least one mutation at an amino acid position selected from the group: S67, T197 and E302. 
     
     
         124 . The mutant M-MLV reverse transcriptase according to  claim 123 , wherein said at least one mutation is selected from the group: S67R, S67N, S67K, T197A, T1975, T197G, E302K, E302R, and E302G.