Recombineering linear DNA that replicate stably in E-coli
Recombineering linear DNA that replicate stably in E-coli
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DOI：
10.1016/j.plasmid.2007.09.002
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发表时间：
2008-01-01
期刊：
影响因子：
2.6
通讯作者：
Narayanah, Kumaran
中科院分区：
文献类型：
--
作者：
Ooi, Yaw-Shin;Warburton, Peter E.;Narayanah, Kumaran
重组工程技术在大肠杆菌中的出现彻底改变了重组DNA分子的构建方式。我们提出了一种新的应用重组工程来线性化DNA，通过用来自噬菌体N15的单个端粒封端来线性化DNA，N15在大肠杆菌中以线性原噬菌体的形式存在。N15端粒酶被重组成环状DNA，并被噬菌体N15原端粒酶分解成单独的端粒。我们通过组装线性BAC来演示这一技术，这些BAC在其宿主菌株E.ColiDH10B中稳定复制。用脉冲场凝胶电泳法进行限制性内切酶图谱分析，证实了BAC的正确线性化。由于端粒的存在，线性BAC DNA可以很容易地用标准的质粒分离方法纯化，并在体内外抵抗RecBCD核酸酶的降解。将含有人β-珠蛋白基因簇的100kb线性BAC导入HT1080细胞，获得了准确剪接的转录本，表明该线性DNA将用于后续的功能研究。这种新的重组工程技术可能对构建用于组装人工染色体和端粒的大型线性结构特别有用，并可能提供一种独特的手段来克隆和研究包含发夹末端的大型线性病毒基因组。(C)2007 Elsevier Inc.保留所有权利。
The advent of recombineering technology in Escherichia coli has revolutionized the way recombinant DNA molecules are constructed. We present a novel application of recombineering to linearize DNA by capping their ends with individual telomeres derived from bacteriophage N15, which exists as a linear prophage in E. coli. The N15 telomerase occupancy site was recombined into circular DNA and resolved into individual telomeres by the phage N15 protelomerase enzyme. We demonstrate this technique by assembling linear BACs that replicate stably in their host strain E coli DH10B. Correct linearization of the BACs was confirmed by restriction mapping using pulsed field gel electrophoresis. The linear BAC DNA can be easily purified using standard plasmid isolation methods and resist degradation from RecBCD nuclease in vitro and in vivo owing to the presence of telomeres. Transfection of a linear 100 kb BAC containing the human beta-globin gene cluster into HT1080 cells produced accurately spliced transcripts, demonstrating that the linear DNA will be useful for subsequent functional studies. This novel recombineering technique may be particularly useful for building large linear constructs for assembling artificial chromosomes with telomeres, and may provide a unique means to clone and study large linear viral genomes that contain hairpin ends. (c) 2007 Elsevier Inc. All rights reserved.