Steiner Paul J, Baumer Zachary T, Whitehead Timothy A
Department of Chemical and Biological Engineering, University of Colorado at Boulder, Colorado, USA.
Bio Protoc. 2020 Aug 5;10(15):e3697. doi: 10.21769/BioProtoc.3697.
Saturation mutagenesis is a fundamental enabling technology for protein engineering and epitope mapping. Nicking mutagenesis (NM) allows the user to rapidly construct libraries of all possible single mutations in a target protein sequence from plasmid DNA in a one-pot procedure. Briefly, one strand of the plasmid DNA is degraded using a nicking restriction endonuclease and exonuclease treatment. Mutagenic primers encoding the desired mutations are annealed to the resulting circular single-stranded DNA, extended with high-fidelity polymerase, and ligated into covalently closed circular DNA by DNA ligase. The heteroduplex DNA is resolved by selective degradation of the template strand. The complementary strand is synthesized and ligated, resulting in a library of mutated covalently closed circular plasmids. It was later shown that because very little primer is used in the procedure, resuspended oligo pools, which normally require amplification before use, can be used directly in the mutagenesis procedure. Because oligo pools can contain tens of thousands of unique oligos, this enables the construction of libraries of tens of thousands of user-defined mutations in a single-pot mutagenesis reaction, which significantly improves the utility of NM as described below. Use of oligo pools afford an economically advantageous approach to mutagenic experiments. First, oligo pool synthesis is much less expensive per nucleotide synthesized than conventional synthesis. Second, a mixed pool may be generated and used for mutagenesis of multiple different genes. To use the same oligo-pool for mutagenesis of a variety of genes, the user must only quantify the fraction of the oligo-pool specific to her mutagenic experiment and adjust the volume and effective concentration of the oligo-pool for use in nicking mutagenesis.
饱和诱变是蛋白质工程和表位作图的一项基本支撑技术。切口诱变（NM）允许用户通过一锅法从质粒DNA快速构建目标蛋白质序列中所有可能的单突变文库。简要地说，使用切口限制性内切酶和核酸外切酶处理降解质粒DNA的一条链。编码所需突变的诱变引物与所得的环状单链DNA退火，用高保真聚合酶延伸，并通过DNA连接酶连接成共价闭合环状DNA。通过模板链的选择性降解来解析异源双链DNA。合成并连接互补链，从而产生突变的共价闭合环状质粒文库。后来发现，由于该过程中使用的引物很少，通常需要在使用前进行扩增的重悬寡核苷酸池可以直接用于诱变过程。由于寡核苷酸池可以包含数万个独特的寡核苷酸，这使得在单锅诱变反应中能够构建数万个用户定义突变的文库，如下所述，这显著提高了NM的实用性。使用寡核苷酸池为诱变实验提供了一种经济上有利的方法。首先，寡核苷酸池合成每合成一个核苷酸的成本比传统合成低得多。其次，可以生成一个混合池并用于多个不同基因的诱变。为了将同一个寡核苷酸池用于多种基因的诱变，用户只需量化其诱变实验特有的寡核苷酸池的比例，并调整寡核苷酸池的体积和有效浓度以用于切口诱变。
