Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase.
Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase.
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DOI：
10.1371/journal.pone.0115318
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Cole RH
中科院分区：
文献类型：
--
作者：
Fu C;Donovan WP;Shikapwashya-Hasser O;Ye X;Cole RH
分子克隆几乎被用于生物学和医学研究的各个方面。我们开发了一种称为热融合的方法，在不使用连接酶的情况下将一个或多个DNA片段有效地克隆到质粒载体中。该方法是定向的，产生无缝连接，并且不依赖于插入物的限制性位点的可用性。片段基于在连接处的17-30 bp的共享同源区组装，这大大简化了构建体设计。热熔合在50°C下以一步单管反应进行1小时，然后冷却至室温。除了用于多片段组装之外，热融合还提供了一种用于克隆含有反向重复序列的DNA片段的高效方法，用于诸如RNAi的应用。总的克隆效率在90- 95%的量级。
Molecular cloning is utilized in nearly every facet of biological and medical research. We have developed a method, termed Hot Fusion, to efficiently clone one or multiple DNA fragments into plasmid vectors without the use of ligase. The method is directional, produces seamless junctions and is not dependent on the availability of restriction sites for inserts. Fragments are assembled based on shared homology regions of 17–30 bp at the junctions, which greatly simplifies the construct design. Hot Fusion is carried out in a one-step, single tube reaction at 50°C for one hour followed by cooling to room temperature. In addition to its utility for multi-fragment assembly Hot Fusion provides a highly efficient method for cloning DNA fragments containing inverted repeats for applications such as RNAi. The overall cloning efficiency is in the order of 90–95%.
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