一种利用 Golden Gate 分子内连接从质粒 DNA 生成用户定义的环状单链 DNA 的方法。
A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation.
机构信息
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado, USA.
出版信息
Biotechnol Bioeng. 2023 Oct;120(10):3057-3066. doi: 10.1002/bit.28471. Epub 2023 Jun 27.
Construction of user-defined long circular single stranded DNA (cssDNA) and linear single stranded DNA (lssDNA) is important for various biotechnological applications. Many current methods for synthesis of these ssDNA molecules do not scale to multikilobase constructs. Here we present a robust methodology for generating user-defined cssDNA employing Golden Gate assembly, a nickase, and exonuclease degradation. Our technique is demonstrated for three plasmids with insert sizes ranging from 2.1 to 3.4 kb, requires no specialized equipment, and can be accomplished in 5 h with a yield of 33%-43% of the theoretical. To produce lssDNA, we evaluated different CRISPR-Cas9 cleavage conditions and reported a 52 ± 8% cleavage efficiency of cssDNA. Thus, our current method does not compete with existing protocols for lssDNA generation. Nevertheless, our protocol can make long, user-defined cssDNA readily available to biotechnology researchers.
构建用户定义的长环形单链 DNA（cssDNA）和线性单链 DNA（lssDNA）对于各种生物技术应用非常重要。目前许多合成这些 ssDNA 分子的方法都无法扩展到多千碱基的构建体。在这里，我们提出了一种使用 Golden Gate 组装、切口酶和核酸外切酶降解来生成用户定义的 cssDNA 的稳健方法。我们的技术已在三个插入大小范围为 2.1 到 3.4kb 的质粒中得到验证，该技术不需要特殊设备，并且可以在 5 小时内完成，理论产量为 33%-43%。为了产生 lssDNA，我们评估了不同的 CRISPR-Cas9 切割条件，并报告了 cssDNA 的 52±8%的切割效率。因此，我们目前的方法并不与现有的 lssDNA 生成协议竞争。尽管如此，我们的方案可以使生物技术研究人员更容易获得长的、用户定义的 cssDNA。
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