Two-step Red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli
Two-step Red-mediated recombination for versatile high-efficiency markerless DNA manipulation in Escherichia coli
复制标题
DOI：
10.2144/000112096
复制
发表时间：
2006-02-01
期刊：
影响因子：
2.7
通讯作者：
Osterrieder, N
中科院分区：
文献类型：
--
作者：
Tischer, BK;von Einem, J;Osterrieder, N
利用pcr扩增的选择性标记进行红色重组是一种成熟的大肠杆菌大分子DNA诱变技术。然而，对于无标记的修饰，包括点突变、缺失，特别是长序列的插入，该系统的有效性和通用性有限。本文描述了一种将红色重组和切割与归巢内切酶1-Sce1结合起来的方法，以实现高效的基于pcr的DNA工程，而不会保留不需要的外源序列。我们将该方法应用于含有所有感染性疱疹病毒克隆的细菌人工染色体（BAC）构建物的修饰，以证明该诱变技术的潜力，该技术可用于插入长序列，如编码区或启动子，引入点突变，无疤痕缺失和插入短序列，如表位标签。该系统被证明是高度可靠和高效的，可适用于各种不同的BAC克隆修饰，是多种应用的基本工具，如转基因动物的产生和基因治疗或疫苗载体的构建。
Red recombination using PCR-amplified selectable markers is a well-established technique for mutagenesis of large DNA molecules in Escherichia coli. The system has limited efficacy and versatility, however for markerless modifications including point mutations, deletions, and particularly insertions of longer sequences. Here e describe a procedure that combines Red recombination and cleavage with the homing endonuclease 1-Sce1 to allow highly efficient, PCR-based DNA engineering without retention of unwanted foreign sequences. We applied the method to modification of bacterial artificial chromosome (BAC) constructs harboring all infectious herpesvirus clone to demonstrate the potential of the mutagenesis technique, which vas used for the insertion of long sequences such as coding regions or promoters, introduction of point mutations, scarless deletions, and insertions of short sequences such as an epitope tag. The system proved to be highly reliable and efficient and call be adapted for a variety of different modifications of BAC clones, which are fundamental tools for applications as diverse as the generation of transgenic animals and the construction of gene therapy or vaccine vectors.