One-step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase

Biotechnology Journal
Ramon KranasterAndreas Marx

Abstract

We describe the cloning and characterization of a mutated thermostable DNA polymerase from Thermus aquaticus (Taq) that exhibits an increased reverse transcriptase activity and is therefore designated for one-step PCR pathogen detection using established real-time detection methods. We demonstrate that this Taq polymerase mutant (Taq M1) has similar PCR sensitivity and nuclease activity as the respective Taq wild-type DNA polymerase. In addition, and in marked contrast to the wild-type, Taq M1 exhibits a significantly increased reverse transcriptase activity especially at high temperatures (>60 degrees C). RNA generally hosts highly stable secondary structure motifs, such as hairpins and G-quadruplexes, which complicate, or in the worst case obviate, reverse transcription (RT). Thus, RT at high temperatures is desired to weaken or melt secondary structure motifs. To demonstrate the ability of Taq M1 for RNA detection of pathogens, we performed TaqMan probe-based diagnostics of Dobrava viruses by one-step RT-PCR. We found similar detection sensitivities compared to commercially available RT-PCR systems without further optimization of reaction parameters, thus making this enzyme highly suitable for any PCR probe-based RNA detecti...Continue Reading

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Citations

Jun 14, 2012·Protein Engineering, Design & Selection : PEDS·Aurimas BaranauskasRemigijus Skirgaila
Nov 13, 2015·Euro Surveillance : Bulletin Européen Sur Les Maladies Transmissibles = European Communicable Disease Bulletin·Oumar FayeAmadou Alpha Sall
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Apr 12, 2016·Analytical Chemistry·Christian D AhrbergPavel Neuzil
Sep 24, 2010·Chembiochem : a European Journal of Chemical Biology·Ramon Kranaster, Andreas Marx
Dec 17, 2010·Chembiochem : a European Journal of Chemical Biology·Feng Du, Zhuo Tang
Aug 21, 2020·Proceedings of the National Academy of Sciences of the United States of America·Woo Suk ChoiWei Yang
Dec 1, 2010·Chembiochem : a European Journal of Chemical Biology·Stanislaw K Jozwiakowski, Bernard A Connolly

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