A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis
A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis
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DOI：
10.21769/bioprotoc.3697
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发表时间：
2020-08-05
期刊：
影响因子：
0.8
通讯作者：
Whitehead, Timothy A.
中科院分区：
文献类型：
--
作者：
Steiner, Paul J.;Baumer, Zachary T.;Whitehead, Timothy A.
饱和诱变是蛋白质工程和表位作图的基本使能技术。切口诱变（NM）允许用户在一锅法中从质粒DNA快速构建靶蛋白序列中所有可能的单突变的文库。简而言之，使用切口限制性内切酶和外切核酸酶处理降解质粒DNA的一条链。编码所需突变的诱变引物与所得环状单链DNA退火，用高保真聚合酶延伸，并通过Taq DNA连接酶连接成共价闭合的环状DNA。异源双链DNA通过模板链的选择性降解来解析。合成并连接互补链，产生突变的共价闭合环状质粒文库。后来表明，由于在该程序中使用非常少的引物，因此通常在使用前需要扩增的重悬寡聚物池可以直接用于诱变程序。由于寡聚物库可以包含数万个独特的寡聚物，这使得能够在单罐诱变反应中构建数万个用户定义的突变的文库，这显著提高了NM的效用，如下所述。首先，与传统合成相比，每个合成的核苷酸的寡聚池合成成本要低得多。第二，可以产生混合池并用于多个不同基因的诱变。为了使用相同的寡聚物池用于多种基因的诱变，用户必须仅定量其诱变实验特异性的寡聚物池的分数，并调整寡聚物池的体积和有效浓度以用于切口诱变。
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 Taq 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.