Facile Assembly of Combinatorial Mutagenesis Libraries Using Nicking Mutagenesis.
Facile Assembly of Combinatorial Mutagenesis Libraries Using Nicking Mutagenesis.
复制标题
DOI：
10.1007/978-1-0716-2152-3_6
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
中科院分区：
文献类型：
--
作者：
组合突变是一种将多个用户定义的突变在一个序列中的指定位置编码的方法。组合诱变文库可用于各种应用，包括评估分子进化的基本问题，酶工程的定向进化工作流程，以及更好地理解生物过程，如抗体亲和力成熟。在这里，我们展示了一种组合突变的方法，利用基于模板的划痕突变和几次修改。我们展示了一个例子，用于生成具有14个突变位置的组合库，总共16,384个库变体，以及用于生成大型的、用户定义的组合库的协议。读者可以使用该协议在两天内创建这样的库。
Combinatorial mutagenesis is a method where multiple user-defined mutations are encoded at defined positions in a sequence. Combinatorial mutagenic libraries can be used in a variety of applications including evaluating fundamental questions about molecular evolution, directed evolution workflows for enzyme engineering, and in better understanding of biological processes like antibody affinity maturation. Here we show a method of combinatorial mutagenesis utilizing the template-based nicking mutagenesis with several modifications. We show an example for generating a combinatorial library with 14 mutated positions, a total of 16,384 library variants, and a protocol for the generation of large, user-defined combinatorial libraries. The reader can use this protocol to create such libraries in two days.