The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes
The univector plasmid-fusion system, a method for rapid construction of recombinant DNA without restriction enzymes
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DOI：
10.1016/s0960-9822(07)00560-x
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发表时间：
1998-12-03
期刊：
影响因子：
9.2
通讯作者：
Elledge, SJ
中科院分区：
文献类型：
--
作者：
Liu, QH;Li, MZ;Elledge, SJ
背景：现代生物学研究高度依赖重组DNA技术。常规的克隆方法是耗时的，缺乏统一性。因此，生物学研究非常需要新技术来快速，系统和统一地操纵目前可从基因组项目中获得的大量基因。分类：我们描述了一系列新的克隆方法，可促进重组DNA分子的快速而系统地构建。中央克隆法被命名为单位质粒融合系统（UPS）。 UPS使用CRE -Lox特定的重组来催化UNIVETOR之间的质粒融合 - 一种包含感兴趣基因的质粒和包含调节信息的宿主向量。融合事件是遗传选择的，并将基因置于新调节元件的控制之下。第二种与UPS相关的方法允许仅将编码序列从单位转移到主机向量。 UPS消除了对亚克隆所需的限制酶，DNA连接酶和许多体外操作的需求，并允许快速构造多种构造以在多种生物体中表达。我们证明，UPS也可以用于将整个库转移到新的媒介中。描述了其他适应性，包括定向PCR克隆和使用同源重组在大肠杆菌中的同源重组的产生3'末端基因融合。结论：这些基于重组的克隆方法共同构成了一种新的综合方法，用于快速有效地产生重组DNA。可用于并行处理大基因集，该特征将促进未来的基因组分析。 （c）当前生物学有限公司ISSN 0960-9822。
Background: Modern biological research is highly dependent upon recombinant DNA technology. Conventional cloning methods are time-consuming and lack uniformity. Thus, biological research is in great need of new techniques to rapidly, systematically and uniformly manipulate the large sets of genes currently available from genome projects.Results: We describe a series of new cloning methods that facilitate the rapid and systematic construction of recombinant DNA molecules. The central cloning method is named the univector plasmid-fusion system (UPS). The UPS uses Cre-lox site-specific recombination to catalyze plasmid fusion between the univector - a plasmid containing the gene of interest - and host vectors containing regulatory information. Fusion events are genetically selected and place the gene under the control of new regulatory elements. A second UPS-related method allows for the precise transfer of coding sequences only from the univector into a host vector. The UPS eliminates the need for restriction enzymes, DNA ligases and many in vitro manipulations required for subcloning, and allows for the rapid construction of multiple constructs for expression in multiple organisms. We demonstrate that UPS can also be used to transfer whole libraries into new vectors. Additional adaptations are described, including directional PCR cloning and the generation of 3' end gene fusions using homologous recombination in Escherichia coli.Conclusions: Together, these recombination-based cloning methods constitute a new comprehensive approach for the rapid and efficient generation of recombinant DNA that can be used for parallel processing of large gene sets, a feature that will facilitate future genomic analysis. (C) Current Biology Ltd ISSN 0960-9822.