聚合酶
分子生物学
DNA聚合酶
生物
水热
RNA聚合酶
DNA
逆转录酶
生物化学
酶
核糖核酸
底漆(化妆品)
实时聚合酶链反应
聚合酶链反应
化学
大肠杆菌
嗜热菌
RNA依赖性RNA聚合酶
核苷酸转移酶
病毒学
过程性
作者
Ramon Kranaster,Matthias Drum,Nicole Engel,Manfred Weidmann,Frank T. Hufert,Andreas Marx
标识
DOI:10.1002/biot.200900200
摘要
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°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 detection method.
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