使用体外生成的蛋白质绘制归巢内切核酸酶切割位点图谱。
Mapping homing endonuclease cleavage sites using in vitro generated protein.
作者信息
Bonocora Richard P, Belfort Marlene
机构信息
New York State Department of Health, Wadsworth Center, Albany, NY, USA.
出版信息
Methods Mol Biol. 2014;1123:55-67. doi: 10.1007/978-1-62703-968-0_4.
Abstract
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.
摘要
绘制核酸内切酶切割位点的精确位置是一种用于描述归巢内切酶功能的基本实验技术。然而，由于错误的DNA切割事件诱导的毒性，这些蛋白质在生物系统中常常难以克隆和过量表达。在本章中，我们概述了在体外成功表达归巢内切酶并将该产物用于核苷酸分辨率切割测定的步骤。
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