| A Method for User-defined Mutagenesis by Integrating Oligo Pool Synthesis Technology with Nicking Mutagenesis
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Author:
Date:
2020-08-05
[Abstract] 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 ...
[摘要] [摘要] 饱和突变是蛋白质工程和表位定位的基础技术。缺口突变（NM）允许用户在一锅程序中快速构建目标蛋白序列中所有可能的单一突变的文库。简言之，质粒DNA的一条链被切痕限制性内切酶和核酸外切酶处理降解。编码所需突变的突变引物退火成环状单链DNA，用高保真聚合酶延伸，并通过taqdna连接酶连接成共价闭合的环状DNA。通过选择性降解模板链来分解异源双链DNA。合成并连接互补链，形成一个共价闭合的环状质粒库。后来的研究表明，由于该过程中使用的引物很少，因此通常在使用前需要扩增的再悬浮寡聚物池可以直接用于诱变过程。因为寡聚体可以包含数以万计的独特寡聚体，这使得在一个单罐突变反应中能够构建数以万计用户定义突变的库，这显著提高了NM的效用，如下所述。
寡聚物的使用为诱变实验提供了一种经济上有利的方法。首先，寡核苷酸池合成比传统合成便宜得多。第二，可以产生一个混合池，用于多个不同基因的诱变。为了使用同一个寡聚体进行多种基因的诱变，使用者只需量化特定于其诱变实验的寡聚体部分，并调整寡聚体的体积和有效浓度，以用于缺口突变。
[背景] 蛋白质功能序列相关性的评价对于蛋白质科学的应用和基础研究具有重要意义。近年来，深度突变扫描（DMS）已经上升到基于蛋白质的研究的前沿（Fowler and ...
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| Plant Sequence Capture Optimised for Illumina Sequencing
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Author:
Date:
2014-07-05
[Abstract] Plant Sequence Capture is used for targeted resequencing of whole exomes (all exons of a genome) of complex genomes e.g. barley and its relatives (Mascher et al., 2013). Sequencing and computing costs are significantly reduced since only the greatly enriched and gene-coding part of the barley genome is targeted, that corresponds to only 1-2% of the entire genome. Thus, applications such as genetic diversity studies and the isolation of single genes (“cloning-by-sequencing”) are greatly facilitated. Here, a protocol is provided describing the construction of shotgun DNA libraries from genomic barley DNA for sequencing on the Illumina HiSeq/MiSeq systems. The shotgun DNA sequencing libraries are hybridized to an oligonucleotide pool (Exome Library) encompassing the whole ...
[摘要] 植物序列捕获用于复杂基因组（例如大麦及其亲属）的整个外显子（基因组的所有外显子）的靶向重测序（Mascher等人，2013）。测序和计算成本显着降低，因为只有大麦基因组的大量富集和基因编码部分被靶向，其仅对应于整个基因组的1-2％。因此，大大促进了诸如遗传多样性研究和单个基因的分离（"通过测序克隆"）的应用。这里，提供了描述来自基因组大麦DNA的Shotgun DNA文库的构建以在Illumina HiSeq/MiSeq系统上测序的方案。鸟枪DNA测序文库与包含大麦整个外显子组的寡核苷酸池（Exome Library）杂交。外显子组库作为包含生物素化探针（Roche/NimbleGen）的液体阵列提供。随后，使用链霉亲和素包被的磁珠对与Exome文库杂交的基因组鸟枪DNA片段进行亲和纯化。捕获的文库被PCR扩增和测序，使用高通量短读序列合成
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