Twin-primer non-enzymatic DNA assembly: an efficient and accurate multi-part DNA assembly method.
Twin-primer non-enzymatic DNA assembly: an efficient and accurate multi-part DNA assembly method.
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DOI：
10.1093/nar/gkx132
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发表时间：
2017-06-20
影响因子：
14.9
通讯作者：
Zhao H
中科院分区：
文献类型：
--
作者：
Liang J;Liu Z;Low XZ;Ang EL;Zhao H
DNA组装是现代合成生物学的基石。尽管有许多可用的方法，但大质粒的无瘢痕多片段组装仍然具有挑战性。此外，即将到来的分子生物自动化浪潮要求我们重新思考如何进行DNA组装。为了简化自动化工作流程并最小化操作员干预，非常需要非酶促组装方法。在这里，我们报告了一个称为双引物组装（TPA）的过程的优化和操作，这是一种将聚合酶链反应扩增片段组装到质粒中而不使用酶的方法。TPA能够以约80%的保真度从10个片段组装7kb质粒，并且以约50%的保真度从5个片段组装31kb质粒。TPA克隆是无瘢痕和序列独立的。即使不使用酶，TPA的性能也与目前可用的一些最好的体外组装方法相当。TPA应该是合成生物学家工具箱中无价的补充。
DNA assembly forms the cornerstone of modern synthetic biology. Despite the numerous available methods, scarless multi-fragment assembly of large plasmids remains challenging. Furthermore, the upcoming wave in molecular biological automation demands a rethinking of how we perform DNA assembly. To streamline automation workflow and minimize operator intervention, a non-enzymatic assembly method is highly desirable. Here, we report the optimization and operationalization of a process called Twin-Primer Assembly (TPA), which is a method to assemble polymerase chain reaction-amplified fragments into a plasmid without the use of enzymes. TPA is capable of assembling a 7 kb plasmid from 10 fragments at ∼80% fidelity and a 31 kb plasmid from five fragments at ∼50% fidelity. TPA cloning is scarless and sequence independent. Even without the use of enzymes, the performance of TPA is on par with some of the best in vitro assembly methods currently available. TPA should be an invaluable addition to a synthetic biologist's toolbox.