Phi29 DNA polymerase is widely used for DNA amplification through rolling-circle replication or multiple displacement amplification. Here, we performed completely in vitro artificial evolution of phi29 DNA polymerase by combining the in vitro compartmentalization and the gene expression-coupled rolling-circle replication of a circular DNA encoding the polymerase. We conducted the experiments in six different conditions composed of three different levels of inhibitor concentrations with two different DNA labeling methods. One of the experiments was performed in our previous study and the other five experiments were newly conducted in this study. Under all conditions, we found several mutations that enhance the rolling-circle amplification by the polymerase when it was expressed in the reconstituted gene expression system. Especially, a combinatorial mutant polymerase (K555T/D570N) exhibits significantly higher rolling-circle activity than the wild type. These highly active mutant polymerases would be useful for various applications.

中文翻译：

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phi29 DNA聚合酶的体外进化通过间隔基因表达和滚环复制来实现。

Phi29 DNA聚合酶广泛用于通过滚环复制或多置换扩增进行DNA扩增。在这里，我们完全进行体外DNA聚合酶的人工进化相结合，在体外分隔和编码聚合酶的环状DNA的基因表达耦合滚环复制。我们用两种不同的DNA标记方法，在由三种不同浓度的抑制剂浓度组成的六种不同条件下进行了实验。其中一项实验是在我们之前的研究中进行的，另外五项实验是在此研究中新进行的。在所有条件下，我们都发现了一些突变，这些突变在重组基因表达系统中表达时增强了聚合酶的滚环扩增。特别是，组合突变型聚合酶（K555T / D570N）的滚环活性明显高于野生型。这些高活性突变体聚合酶可用于各种应用。