| Abstract [eng] |
Complementary DNA synthesis is one of the most used molecular biology methods which is performed by reverse transcriptases. Synthesized complementary DNA can be applied in cloning, gene expression and sequencing procedures. One of such cDNA producing enzymes is Moloney leukemia virus reverse transcriptase which is distinguished by a high level of synthesis activity. Significant application of Moloney reverse transcriptase motivates to create better performing mutants that could have a higher synthesis activity, processivity and an increased resistance to inhibitors. Therefore identified reverse transcriptase mutants with desirable properties could improve variety of molecular biology experiments. In previous experiments 12 unique mutations that could improve Moloney leukemia virus reverse transcriptase thermostability, resistance to inhibitors and other properties were identified. The aim of the study was to examine previously developed 12 M-MuLV RT mutants and select the best performing mutations for further multiple mutant construction. During this work, the effect of these 12 mutations on reverse transcriptase thermostability, resistance to inhibitors and sensitivity in one-step RT-qPCR was identified. Mutations that had the best positive impact on the features of reverse transcriptase were selected for further double and triple single-point mutant construction. During the research multiple mutants of reverse transcriptase were purified and it was shown that the collection of several mutations could have a more positive influence on enzyme features. |
