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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Increasing cloning possibilities using artificial zinc finger nucleases
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Increasing cloning possibilities using artificial zinc finger nucleases

机译：使用人工锌指核酸酶增加克隆的可能性

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摘要

The ability to accurately digest and ligate DNA molecules of different origins is fundamental to modern recombinant DNA research. Only a handful of enzymes are capable of recognizing and cleaving novel and long DNA sequences, however. The slow evolution and engineering of new restriction enzymes calls for alternative strategies to design novel and unique restriction enzymes capable of binding and digesting specific long DNA sequences. Here we report on the use of zinc finger nucleases (ZFNs)-hybrid synthetic restriction enzymes that can be specifically designed to bind and cleave long DNA sequences-for the purpose of DNA recombination. We show that novel ZFNs can be designed for the digestion of specific sequences and can be expressed and used for cloning purposes. We also demonstrate the power of ZFNs in DNA cloning by custom-cloning a target DNA sequence and assembling dual-expression cassettes on a single target plasmid, a task that rarely can be achieved using type-II restriction enzymes. We demonstrate the flexibility of ZFN design and the ability to shuffle monomers of different ZFNs for the digestion of compatible recognition sites through ligation of compatible ends and their cleavage by heterodimer ZFNs. Of no less importance, we show that ZFNs can be designed to recognize and cleave existing DNA sequences for the custom-cloning of native target DNA molecules.
机译：准确消化和连接不同来源的DNA分子的能力是现代重组DNA研究的基础。但是，只有少数几种酶能够识别和切割新颖的长DNA序列。新的限制酶的缓慢发展和工程化要求采用替代策略来设计能够结合和消化特定长DNA序列的新颖独特的限制酶。在这里，我们报告了锌指核酸酶（ZFNs）-杂交合成限制酶的使用，该酶可以专门设计用于结合和切割长DNA序列-以实现DNA重组。我们表明新型ZFNs可以设计用于特定序列的消化，并可以表达并用于克隆目的。我们还展示了通过自定义克隆目标DNA序列并在单个目标质粒上组装双表达盒，ZFN在DNA克隆中的功能，使用II型限制酶很少能实现这一任务。我们展示了ZFN设计的灵活性，以及​​通过连接兼容末端并被异二聚体ZFN裂解来改组不同ZFN的单体以消化兼容识别位点的能力。同样重要的是，我们显示ZFN可以被设计为识别和切割现有的DNA序列，用于定制克隆天然目标DNA分子。

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