Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
Biotechnol J. 2010 Feb;5(2):224-31. doi: 10.1002/biot.200900200.
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 detection method.
我们描述了一种来自水生栖热菌（Taq）的突变耐热 DNA 聚合酶的克隆和特性，该聚合酶表现出增强的逆转录酶活性，因此被指定用于使用现有的实时检测方法进行一步式 PCR 病原体检测。我们证明，这种 Taq 聚合酶突变体（Taq M1）与相应的 Taq 野生型 DNA 聚合酶具有相似的 PCR 灵敏度和核酸酶活性。此外，与野生型相比，Taq M1 表现出明显增强的逆转录酶活性，尤其是在高温（>60°C）下。RNA 通常具有高度稳定的二级结构基序，例如发夹和 G-四联体，这会使逆转录（RT）变得复杂，或者在最坏的情况下使其无效。因此，需要在高温下进行 RT，以削弱或溶解二级结构基序。为了证明 Taq M1 用于检测病原体 RNA 的能力，我们通过一步 RT-PCR 对 Dobrava 病毒进行了 TaqMan 探针为基础的诊断。与没有进一步优化反应参数的商业 RT-PCR 系统相比，我们发现了相似的检测灵敏度，因此这种酶非常适合任何基于 PCR 探针的 RNA 检测方法。
