Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.
Synthesis and cell-free cloning of DNA libraries using programmable microfluidics.
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DOI：
10.1093/nar/gkv1087
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发表时间：
2016-02-29
影响因子：
14.9
通讯作者：
Shapiro E
中科院分区：
文献类型：
--
作者：
Ben Yehezkel T;Rival A;Raz O;Cohen R;Marx Z;Camara M;Dubern JF;Koch B;Heeb S;Krasnogor N;Delattre C;Shapiro E
微流体技术可能会通过解决与生成新的遗传结构相关的几个基本限制来彻底改变我们编写合成DNA的能力。在这里，我们报告了使用可编程数字微流体在亚微升反应液滴中首次从头合成和无细胞克隆定制DNA文库。具体而言，我们开发了可编程有序聚合（POP）、DNA的微流体组合组装（M-CAD）和微流体体外克隆（MIC），并将它们分别应用于基因的从头合成、组合组装和无细胞克隆。这些方法的概念验证是通过编程一个自主的微流体系统来构建和克隆酵母核糖体结合位点和细菌天青蛋白的文库，然后在单个液滴中检索并验证。以自主方式快速和稳健地产生设计者DNA分子的能力应该在生物研究和开发中具有广泛的应用。
Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development.