Nils Rutschke

• Reverse transcriptase (RT) is an indispensable enzyme for real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR), a standard method in molecular diagnostics for detection and quantification of defined RNA molecules. The prevention of non-specific products due to elongation of misprimed oligonucleotides by the enzyme at temperatures beneath the specific annealing temperature is one of the biggest challenges in real-time RT-PCR. A reduction of non-specific products could be achieved by the optimization of the reaction conditions and/or by the use of so called hot start enzymes. Hot start enzymes are inactive at lower temperatures, when mispriming occurs and activated at higher temperatures, appropriate for specific annealing of the primers (short oligonucleotides) and their extension. Different hot start mechanisms are described for the thermostable DNA-dependent DNA polymerase (the key enzyme in PCR), but for the RT is still missing. The aim of this PhD study is to investigate the potential for a hot start moloney murine leukemia virus (MMLV) RT variant with increased thermostability in real-time RT-PCR, leading to an improved performance of this technique. Therefore, different strategies are followed: temperature-dependent inactivation of RT in real-time RT-PCR by (1) aptamers or antibodies and (2) temperature sensitive encapsulation. Furthermore, the improvement of the thermostability of the enzyme due to mutations, reaction buffer optimization or both, to gain a higher flexibility for the hot start approaches. In the study a hot start RT for real-time RT-PCR by temperature-dependent inactivation with aptamers was realised, which led to an increase in analytical sensitivity of a real-time RT-PCR assay. Enhanced thermostability of a MMLV RT variant and improved real-time RT-PCR performance, compared to a wild-type MMLV RT was realized.