Mapping homing endonuclease cleavage sites using in vitro generated protein.
Mapping homing endonuclease cleavage sites using in vitro generated protein.
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DOI：
10.1007/978-1-62703-968-0_4
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发表时间：
2014
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影响因子：
--
通讯作者：
Belfort, Marlene
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文献类型：
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作者：
Bonocora, Richard P;Belfort, Marlene
精确定位内切酶的位置是描述核酸内切酶功能的基本实验技术。然而，由于虚假的DNA裂解事件引起的毒性，这些蛋白质经常在生物系统中抵抗克隆和过度表达。在这一章中，我们概述了在体外成功表达归巢内切酶的步骤，并将该产物用于核苷酸拆分切割分析。
Mapping the precise position of endonucleolytic cleavage sites is a fundamental experimental technique used to describe the function of a homing endonuclease. However, these proteins are often recalcitrant to cloning and over-expression in biological systems because of toxicity induced by spurious DNA cleavage events. In this chapter we outline the steps to successfully express a homing endonuclease in vitro and use this product in nucleotide-resolution cleavage assays.