Mishin Andrew A, Groth Tobin, Green Richard E, Troll Christopher J
Claret Bioscience LLC, 100 Enterprise Way, Suite A102, Scotts Valley, CA 95066, United States.
Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, United States.
Synth Biol (Oxf). 2024 Dec 13;9(1):ysae019. doi: 10.1093/synbio/ysae019. eCollection 2024.
In this study, we introduce a new method for oligonucleotide fragment assembly. Unlike polymerase chain assembly and ligase chain assembly that rely on short, highly purified oligonucleotides, our method, named , uses a one-tube, splint-driven assembly reaction. Splynthesis connects standard-desalted "contig" oligos (∼150 nt in length) via shorter "splint" oligos harboring 5' and 3' blocking modifications to prevent off-target ligation and amplification events. We demonstrate the method to assemble a 741-bp gene fragment. We verify the assembled polymerase chain reaction product using standard molecular biology techniques, as well as long-read Oxford Nanopore sequencing, and confirm that the product is cloneable via molecular means, as well as Sanger sequencing. This approach is applicable for synthetic biology, directed evolution, functional protein assays, and potentially even splint-based ligase chain reaction assays.
在本研究中，我们介绍了一种用于寡核苷酸片段组装的新方法。与依赖短的、高度纯化的寡核苷酸的聚合酶链组装和连接酶链组装不同，我们的方法名为Splynthesis，它使用单管夹板驱动的组装反应。Splynthesis通过带有5'和3'封闭修饰的较短“夹板”寡核苷酸连接标准脱盐的“重叠群”寡核苷酸（长度约150 nt），以防止非靶向连接和扩增事件。我们展示了Splynthesis方法组装一个741 bp基因片段的过程。我们使用标准分子生物学技术以及长读长牛津纳米孔测序验证了组装的聚合酶链反应产物，并确认该产物可通过分子手段以及桑格测序进行克隆。这种方法适用于合成生物学、定向进化、功能蛋白测定，甚至可能适用于基于夹板的连接酶链反应测定。
