Thermostable reverse transcriptases and uses thereof
View Patent ↗The present invention is in the fields of molecular and cellular biology. The invention is generally related to reverse transcriptase enzymes and methods for the reverse transcription of nucleic acid molecules, especially messenger RNA molecules. Specifically, the invention relates to reverse transcriptase enzymes which have been mutated or modified to increase thermostability, decrease terminal deoxynucleotidyl transferase activity, and/or increase fidelity, and to methods of producing, amplifying or sequencing nucleic acid molecules (particularly cDNA molecules) using these reverse transcriptase enzymes or compositions. The invention also relates to nucleic acid molecules produced by these methods and to the use of such nucleic acid molecules to produce desired polypeptides. The invention also concerns kits comprising such enzymes or compositions.
1. A mutant M-MLV reverse transcriptase having at least 90% sequence identity to SEQ ID NO:2 and having a first amino acid substitution at position T330 and at least one or more additional amino acid substitutions in the amino acid sequence corresponding to positions 34-716of SEQ ID NO:2, wherein amino acid number 1 of said mutant M-MLV reverse transcriptase corresponds to the threonine at position 34 of SEQ ID NO:2 and the at least one or more additional amino acid substitutions are selected from the group consisting of P127, and G138;
wherein said mutant M-MLV reverse transcriptase shows increased thermostability compared to a M-MLV reverse transcriptase comprising amino acids 34-716 of SEQ ID NO:2 having no amino acid substitutions.
2. The mutant M-MLV reverse transcriptase of claim 1 , wherein said position corresponding to amino acid T330 of SEQ ID NO:2 is substituted with P.
3. The mutant M-MLV reverse transcriptase of claim 1 , wherein said position corresponding to amino acid P127 of SEQ ID NO:2 is substituted with T or H.
4. The mutant M-MLV reverse transcriptase of claim 1 , wherein said position corresponding to amino acid G138 of SEQ ID NO:2 is substituted with R.