Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method
Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method
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DOI：
10.1016/j.ab.2010.07.011
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发表时间：
2010-11-15
影响因子：
2.9
通讯作者：
Schwaneberg, Ulrich
中科院分区：
文献类型：
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作者：
Blanusa, Milan;Schenk, Alexander;Schwaneberg, Ulrich
为了克服限制性克隆存在的转化效率低、载体背景高等问题，人们开发了许多不依赖连接酶的克隆方法。这些方法中的大多数仍然是基于酶的，需要耗时的孵育和多个纯化步骤，和/或在处理中可能具有低稳健性。因此，为了建立一种稳健的无酶/连接酶方法，我们开发了基于硫代磷酸酯的连接酶非依赖性基因克隆（PLICing）方法，该方法基于碘/乙醇溶液中硫代磷酸酯键的化学裂解反应。在优化聚合酶链反应（PCR）和DNA切割条件后，PLICing在操作和转化效率方面与所有商业化方法具有竞争力。此外，PLICing是绝对序列独立的，并且超过了关于克隆效率的其他概念，因为240个分析的克隆中没有一个显示出三种不同模型基因的任何再连接事件。开发的快速PLICing协议不需要任何纯化步骤，可以在10分钟内完成。由于其稳健性，可靠性和简单性，PLICing应该被证明是一个真正的替代其他成熟的克隆技术。(C)2010年爱思唯尔公司All rights reserved.
Many ligase-independent cloning methods have been developed to overcome problems of standard restriction cloning such as low transformation efficiency and high background of vector with no insert. Most of these methods are still enzyme based, require time-consuming incubation and multiple purification steps, and/or might have a low robustness in handling. Thus, with the aim to establish a robust enzyme/ligase-free method, we developed the phosphorothioate-based ligase-independent gene cloning (PLICing) method, which is based on a chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution. After optimization of polymerase chain reaction (PCR) and DNA cleavage conditions, PLICing performs competitively with all commercialized methods in terms of handling and transformation efficiency. In addition, PLICing is absolutely sequence independent and surpasses other concepts regarding cloning efficiency given that none of the 240 analyzed clones showed any religation event for three different model genes. A developed fast PLICing protocol does not require any purification step and can be completed within 10 min. Due to its robustness, reliability, and simplicity, PLICing should prove to be a true alternative to other well-established cloning techniques. (C) 2010 Elsevier Inc. All rights reserved.