Personalized medicine and advanced diagnostic tools based on RNA analysis are focusing on fast and direct One-Step RT-PCR assays. First strand complementary DNA (cDNA) synthesized by the reverse transcriptase (RT) is exponentially amplified in the end-point or real-time PCR. Even a minor discrepancy in PCR conditions would result in big deviations during the data analysis. Thus, One-Step RT-PCR composition is typically based on the PCR buffer. In this study, we have used compartmentalized ribosome display technique for in vitro evolution of the Moloney Murine Leukemia Virus reverse transcriptase (M-MuLV RT) that would be able to perform efficient full-length cDNA synthesis in PCR buffer optimized for Thermus aquaticus DNA polymerase. The most frequent mutations found in a selected library were analyzed. Aside from the mutations, which switch off RNase H activity of RT and are beneficial for the full-length cDNA synthesis, we have identified several mutations in the active center of the enzyme (Q221R and V223A/M), which result in 4-5-fold decrease of Km for dNTPs (<0.2 mM). The selected mutations are in surprising agreement with the natural evolution process because they transformed the active center from the oncoretroviral M-MuLV RT-type to the lenitiviral enzyme-type. We believe that this was the major and essential phenotypic adjustment required to perform fast and efficient cDNA synthesis in PCR buffer at 0.2-mM concentration of each dNTP.

中文翻译：

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降低dNTP的Km是在一步式RT-PCR分析中执行有效cDNA合成所需的必需M-MuLV逆转录酶。

基于RNA分析的个性化医学和先进的诊断工具致力于快速直接的一步式RT-PCR分析。由逆转录酶（RT）合成的第一链互补DNA（cDNA）在终点或实时PCR中被指数扩增。即使PCR条件略有差异，在数据分析过程中也会导致较大的偏差。因此，一步式RT-PCR组合物通常基于PCR缓冲液。在这项研究中，我们已使用区室化的核糖体展示技术对莫洛尼鼠白血病病毒逆转录酶（M-MuLV RT）进行了体外进化，该技术将能够在针对Thermus aquaticus DNA聚合酶优化的PCR缓冲液中进行有效的全长cDNA合成。 。分析在选定文库中发现的最频繁的突变。除了突变，从而关闭了RT的RNase H活性并有利于全长cDNA合成，我们在酶的活性中心（Q221R和V223A / M）中发现了几个突变，这些突变导致Km降低4-5倍对于dNTP（<0.2 mM）。所选的突变与自然进化过程令人惊讶地一致，因为它们将活性中心从核心病毒M-MuLV RT型转变为慢病毒酶型。我们相信，这是在每个dNTP浓度为0.2-mM的PCR缓冲液中进行快速有效的cDNA合成所需的主要和必要的表型调节。2 mM）。所选的突变与自然进化过程令人惊讶地一致，因为它们将活性中心从核心病毒M-MuLV RT型转变为慢病毒酶型。我们相信，这是在每个dNTP浓度为0.2-mM的PCR缓冲液中进行快速有效的cDNA合成所需的主要和必要的表型调节。2 mM）。所选的突变与自然进化过程令人惊讶地一致，因为它们将活性中心从核心病毒M-MuLV RT型转变为慢病毒酶型。我们相信，这是在每个dNTP浓度为0.2-mM的PCR缓冲液中进行快速有效的cDNA合成所需的主要和必要的表型调节。