Rapid and Reliable DNA Assembly via Ligase Cycling Reaction
Rapid and Reliable DNA Assembly via Ligase Cycling Reaction
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DOI：
10.1021/sb4001992
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发表时间：
2014-02-01
影响因子：
4.7
通讯作者：
Chandran, Sunil S.
中科院分区：
文献类型：
--
作者：
de Kok, Stefan;Stanton, Leslie H.;Chandran, Sunil S.
将DNA部分组装成DNA构建体是合成生物学新兴领域的基础技术。高效的DNA组装方法对于生物制造设施中的高通量自动化DNA组装特别重要，因此我们研究了通过连接酶循环反应（LCR）的一步无痕DNA组装。LCR组装使用与相邻DNA部分的末端互补的单链桥接寡核苷酸、用于连接DNA主链的热稳定连接酶以及多个变性-退火-连接温度循环来组装复杂的DNA构建体。提高了LCR装配效率。4-折叠使用设计的优化实验和响应曲面法。在这些优化的条件下，LCR能够一步组装多达20个DNA部分和多达20 kb的DNA构建体，具有非常少的单核苷酸多态性（
Assembly of DNA parts into DNA constructs is a foundational technology in the emerging field of synthetic biology. An efficient DNA assembly method is particularly important for high-throughput, automated DNA assembly in biofabrication facilities and therefore we investigated one-step, scarless DNA assembly via ligase cycling reaction (LCR). LCR assembly uses single-stranded bridging oligos complementary to the ends of neighboring DNA parts, a thermostable ligase to join DNA backbones, and multiple denaturation-annealing-ligation temperature cycles to assemble complex DNA constructs. The efficiency of LCR assembly was improved ca. 4-fold using designed optimization experiments and response surface methodology. Under these optimized conditions, LCR enabled one-step assembly of up to 20 DNA parts and up to 20 kb DNA constructs with very few single-nucleotide polymorphisms (