High fidelity reverse transcriptases and the uses thereof
View Patent ↗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. An MMLV reverse transcriptase which has been modified or mutated to decrease or eliminate terminal deoxynucleotidyl transferase activity, the reverse transcriptase comprising a polymerase domain having RNA-dependent DNA polymerase activity and a substitution in the amino acid sequence of the wild type MMLV polymerase domain within SEQ ID NO: 6, wherein amino acid number 1 of SEQ ID NO: 6 is the threonine following the initial methionine, and wherein said reverse transcriptase comprises at least one mutation at an amino acid position selected from the group consisting of Tyr133, Thr197, and Phe309.
2. The MMLV reverse transcriptase of claim 1 , wherein Tyr133 is replaced with an alanine.
3. The MMLV reverse transcriptase of claim 1 , wherein Thr197 is replaced with a glutamic acid.
4. The MMLV reverse transcriptase of claim 1 , wherein Phe309 is replaced with an asparagine.
5. The MMLV reverse transcriptase of claim 1 , wherein the reverse transcriptase has reduced RNase H activity.
6. The MMLV reverse transcriptase of claim 5 , wherein the reverse transcriptase comprises one or more mutations selected from the group consisting of Asp524Gly, Asp583Asn and Glu562Gln.