Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 71, 9052 Ghent, Belgium.
VIB Center for Plant Systems Biology, Technologiepark 71, 9052 Ghent, Belgium.
ACS Synth Biol. 2022 Jun 17;11(6):2214-2220. doi: 10.1021/acssynbio.2c00072. Epub 2022 Jun 8.
The assembly of DNA parts is a critical aspect of contemporary biological research. Gibson assembly and Golden Gate cloning are two popular options. Here, we explore the use of single stranded DNA oligos with Gibson assembly to augment Golden Gate cloning workflows in a process called "oligo stitching". Our results show that oligo stitching can efficiently convert Golden Gate parts between different assembly standards and directly assemble incompatible Golden Gate parts without PCR amplification. Building on previous reports, we show that it can also be used to assemble sequences. As a final application, we show that restriction enzyme recognition sites can be removed from plasmids and utilize the same concept to perform saturation mutagenesis. Given oligo stitching's versatility and high efficiency, we expect that it will be a useful addition to the molecular biologist's toolbox.
DNA 片段的组装是当代生物学研究的一个关键方面。 Gibson 组装和 Golden Gate 克隆是两种流行的选择。在这里，我们探索了使用单链 DNA 寡核苷酸与 Gibson 组装来增强 Golden Gate 克隆工作流程的方法，这个过程称为“寡核苷酸拼接”。我们的结果表明，寡核苷酸拼接可以有效地在不同的组装标准之间转换 Golden Gate 片段，并且无需 PCR 扩增即可直接组装不兼容的 Golden Gate 片段。在此基础上，我们还展示了它可以用于组装序列。作为最后的应用，我们展示了可以从质粒中去除限制酶识别位点，并利用相同的概念进行饱和突变。鉴于寡核苷酸拼接的多功能性和高效率，我们预计它将成为分子生物学家工具包的有用补充。
