Recombinant reverse transcriptases

Abstract

The present invention relates to a gene that encodes a hyperactive reverse transcriptase having DNA polymerase activity and substantially reduced RNase H activity, vectors containing the gene and host cells transformed with the invention. The present invention also includes a method of producing the hyperactive reverse transcriptase, producing cDNA from mRNA using the reverse transcriptase of the invention, kits and assay templates made using the hyperactive reverse transcriptase.

1 .- 35 . (canceled) 
     
     
         36 . An isolated protein comprising DNA polymerase activity and substantially reduced RNase H activity comprising one or more mutations in the processivity domain and/or in the nucleotide selection domain. 
     
     
         37 . The protein of  claim 36 , wherein the protein produces a yield of greater than about 1, 5, 7, 10, 12, 15, 25 μg of an aRNA from 100 ng of template RNA in a single amplification reaction. 
     
     
         38 . The protein of  claim 36 , wherein the protein produces an aRNA yield of greater than about 20% as compared to an equivalent wild-type Reverse Transcriptase enzyme. 
     
     
         39 . The protein of  claim 36 , wherein the protein produces a yield of greater than about 5 or 10 μg of an aRNA from 10 pg of template RNA after a two-round amplification reaction. 
     
     
         40 . The protein of  claim 36 , wherein the protein produces a cDNA greater than about 6, 9 or even 11 kilobases in a single cDNA synthesis reaction. 
     
     
         41 . The protein of  claim 36 , wherein the protein produces a cDNA greater than about 6 to about 15 kilobases in a single cDNA synthesis reaction. 
     
     
         42 . The protein of  claim 36 , wherein the protein produces a cDNA greater than about 15 kilobases in a single cDNA synthesis reaction. 
     
     
         43 . The protein of  claim 36 , wherein the DNA polymerase activity is greater than about 200 Units per microgram. 
     
     
         44 . The protein of  claim 36 , wherein the DNA polymerase activity is between about 0.1 and 300 Units per microgram. 
     
     
         45 . The protein of  claim 36 , wherein the RNase H activity is between about 0.1 and about 25 percent of the wild-type RNase H activity. 
     
     
         46 .- 61 . (canceled) 
     
     
         62 . An isolated and purified nucleic acid comprising a hyperactive reverse transcriptase with a mutation in the processivity domain and/or in the nucleotide selection domain. 
     
     
         63 . The nucleic acid of  claim 62 , wherein the hyperactive reverse transcriptase comprises a mutation that corresponds to and includes an H638G mutation of the MMLV-RT. 
     
     
         64 . The nucleic acid of  claim 62 , wherein the hyperactive reverse transcriptase comprises a hyperactive reverse transcriptase further comprising an F155Y mutation. 
     
     
         65 . The nucleic acid of  claim 62 , wherein the hyperactive reverse transcriptase comprises a hyperactive reverse transcriptase further comprising an F155Y mutation and an H638G mutation. 
     
     
         66 . The nucleic acid of  claim 62 , wherein the nucleic acid of SEQ ID No.: 1 further comprises a nucleic acid segment encoding a leader sequence. 
     
     
         67 . The nucleic acid of  claim 62 , wherein the nucleic acid of SEQ ID NO.: 1 further comprises a nucleic acid segment encoding a protein segment other than the hyperactive reverse transcriptase. 
     
     
         68 .- 85 . (canceled) 
     
     
         86 . A kit for nucleic acid synthesis, comprising, in a suitable container:
 a hyperactive reverse transcriptase; and   a reaction solution for the reverse transcriptase.   
     
     
         87 . The kit of  claim 86 , further comprising an insert that comprises information for using the reverse transcriptase. 
     
     
         88 . The kit of  claim 86 , wherein the reaction solution comprises a reverse transcriptase reaction buffer. 
     
     
         89 . The kit of  claim 86 , further comprising a primer. 
     
     
         90 . The kit of  claim 86 , wherein the reaction solution comprises a reverse transcriptase buffer. 
     
     
         91 . The kit of  claim 86 , wherein the reaction solution comprises a PCR buffer. 
     
     
         92 . The kit of  claim 86 , further comprising a mix of nucleotides. 
     
     
         93 . The kit of  claim 86 , further comprising containers comprising individual nucleotides. 
     
     
         94 . The kit of  claim 86 , wherein the reaction solution comprises a buffer for in vitro transcription. 
     
     
         95 . The kit of  claim 86 , further comprising a template purification column. 
     
     
         96 . The kit of  claim 86 , further comprising magnetic particles suitable for nucleic acid purification. 
     
     
         97 .- 128 . (canceled)