Most DNA polymerase libraries sample unknown portions of mutational space and are constrained by the limitations of random mutagenesis. Here we describe a programmed allelic mutagenesis (PAM) strategy to comprehensively evaluate all possible single-point mutations in the entire catalytic domain of a replicative DNA polymerase. By applying the PAM strategy with ultrafast high-throughput screening, we show how DNA polymerases can be mapped for allelic mutations that exhibit enhanced activity for unnatural nucleic acid substrates. We suggest that comprehensive missense mutational scans may aid the discovery of specificity determining residues that are necessary for reprogramming the biological functions of natural DNA polymerases.

中文翻译：

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具有单个氨基酸分辨率的DNA聚合酶的程序化等位基因诱变。

大多数DNA聚合酶文库对突变空间的未知部分进行采样，并受到随机诱变的限制。在这里，我们描述了一种程序化的等位基因诱变（PAM）策略，以全面评估复制性DNA聚合酶整个催化域中所有可能的单点突变。通过应用具有超快速高通量筛选的PAM策略，我们展示了DNA聚合酶如何定位于对非天然核酸底物表现出增强活性的等位基因突变。我们建议全面的错义突变扫描可能有助于发现特异性决定残基的发现，这些残基对于重新编程天然DNA聚合酶的生物学功能是必需的。