At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants—urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.

中文翻译：

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具有改进的合成能力和抑制剂耐受性的Bst样Gss聚合酶衍生物

目前，医学诊断的实际趋势之一是针对实际应用的方法的发展，例如即时检验，POCT或研究工具，例如全基因组扩增，WGA。所有技术均基于使用特定的DNA聚合酶，这些酶具有链置换活性，高合成加工性，保真度，最重要的是对污染物的耐受性，这些污染物来自分析的生物样品或在纯化程序中收集到。在这里，我们基于Geobacillus sp。的DNA聚合酶I的催化结构域设计了一套融合酶。777与DNA的DNA结合结构域连接酶激烈热abyssi和Sto7d蛋白从硫化叶菌，与Sso7d类似。设计的嵌合DNA聚合酶DBD-Gss，Sto-Gss和Gss-Sto具有与天然Gss相同水平的热稳定性，热转移酶活性和保真度。但是，合成能力提高到3倍，导致WGA中DNA产物的4倍之多。DNA结合蛋白的连接增强了嵌合聚合酶在环介导的等温扩增中对几种最常见的DNA样品污染物（尿素和全血，肝素，乙二胺四乙酸，NaCl，乙醇）的抑制剂耐受性。因此，由于增加的生产率和抑制剂耐受性，嵌合的Bst样Gss聚合酶将是用于WGA和POCT的有前途的工具。