High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips.
High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips.
复制标题
使用 DNA 芯片上合成的寡核苷酸进行高通量定点诱变。
DOI：
10.2144/05393st04
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发表时间：
2005
期刊：
影响因子：
2.7
通讯作者：
M. Delcourt
中科院分区：
文献类型：
--
作者：
D. Saboulard;V. Dugas;M. Jaber;J. Broutin;É. Souteyrand;J. Sylvestre;M. Delcourt
定点诱变极大地帮助研究人员了解编码序列中特定残基的精确作用，并生成获得新特性的蛋白质变体。当今对更完整的蛋白质功能制图的需求以及选择和筛选技术的进步要求定点诱变适应高通量应用。我们在这里描述了第一代使用在 DNA 芯片上合成的寡核苷酸混合物的单和多定点突变体文库。我们使用人类白介素 15 (IL15) 基因作为模型，其中 37 个密码子同时被 8 个可能的密码子中的任何一个取代。对 96 个克隆进行了测序，在整个基因长度上表现出广泛的靶向替换，没有不需要的突变。使用此类寡核苷酸库生成的文库能够从任何基因简单、快速且经济高效地生成大量定制的遗传多样性，从而为指导蛋白质的体外进化开辟了新的视角。
Site-directed mutagenesis has greatly helped researchers both to understand the precise role of specific residues in coding sequences and to generate variants of proteins that have acquired new characteristics. Today's demands for more complete functional cartographies of proteins and advances in selection and screening technologies require that site-directed mutagenesis be adapted for high-throughput applications. We describe here the first generation of a library of single and multiple site-directed mutants using a mixture of oligonucleotides synthesized on DNA chips. We have used the human interleukin 15 (IL15) gene as a model, of which 37 codons were simultaneously targeted for substitution by any of eight possible codons. Ninety-six clones were sequenced, exhibiting a broad spectrum of targeted substitutions over the whole gene length with no unwanted mutations. Libraries produced using such pools of oligonucleotides open new perspectives to direct the evolution of proteins in vitro, by enabling the simple, rapid, and cost-effective generation of large tailor-made genetic diversities from any gene.
影响因子：
14.9
作者：
Chen, ZD;Ruffner, DE
通讯作者：
Ruffner, DE