Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: Functional analysis of multi-segment expression clones
Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: Functional analysis of multi-segment expression clones
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DOI：
10.1101/gr.2512204
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发表时间：
2004-10-01
期刊：
影响因子：
7
通讯作者：
Brasch, MA
中科院分区：
文献类型：
--
作者：
Cheo, DL;Titus, SA;Brasch, MA
克隆和操纵DNA片段的能力是实现基因、蛋白质和调节元件的表达、筛选和功能表征的分子方法的核心。我们之前描述了一种新技术的发展，该技术利用体外特定部位的重组为高通量克隆和转移DNA片段提供一个强大而灵活的平台。通过使用具有独特特异性的重组位点的扩展谱系，我们扩展了这项技术，使多个DNA片段能够以预定的顺序、方向和阅读框架在体外高效组装和协同克隆到载体骨架中。这种方法的效率和灵活性使功能元素的集合能够以组合的方式生成和混合，以用于众多多段构造的并行组装。组装的构建体可以通过引导定义的片段与交替的DNA片段的交换来进一步操纵。在这份报告中，我们论证了该技术在融合蛋白的产生、启动子与基因的连接以及多个蛋白结构域的组装方面的可行性。这项技术对细胞和蛋白质工程、多结构域蛋白的表达和基因功能分析具有广泛的意义。
The ability to clone and manipulate DNA segments is central to molecular methods that enable expression, screening, and functional characterization of genes, proteins, and regulatory elements. We previously described the development of a novel technology that utilizes in vitro site-specific recombination to provide a robust and flexible platform for high-throughput cloning and transfer of DNA segments. By using an expanded repertoire of recombination sites with unique specificities, we have extended the technology to enable the high-efficiency in vitro assembly and concerted cloning of multiple DNA segments into a vector backbone in a predefined order, orientation, and reading frame. The efficiency and flexibility of this approach enables collections of functional elements to be generated and mixed in a combinatorial fashion for the parallel assembly of numerous multi-segment constructs. The assembled constructs can be further manipulated by directing exchange of defined segments wits alternate DNA segments. In this report, we demonstrate feasibility of the technology and application to the generation of fusion proteins, the linkage of promoters to genes, and the assembly of multiple protein domains. The technology has broad implications for cell and protein engineering, the expression of multidomain proteins, and gene function analysis.