General method for plasmid construction using homologous recombination
General method for plasmid construction using homologous recombination
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DOI：
10.2144/99261rr02
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发表时间：
1999-01-01
期刊：
影响因子：
2.7
通讯作者：
Sexson, SL
中科院分区：
文献类型：
--
作者：
Raymond, CK;Pownder, TA;Sexson, SL
我们描述了一种使用酵母和超越酵母特异性研究应用的质粒组装的通用方法。该技术利用酿酒酵母的同源重组、双链断裂修复途径连接DNA片段。利用合成的双链“重组连接子”将DNA片段“亚克隆”成质粒，效率为80%左右。给出了DNA浓度和重叠长度对重组效率影响的定量数据。使用一个简单的程序，质粒从酵母中穿梭到大肠杆菌中，用于随后的筛选和大规模质粒制备。这种简单的质粒构建方法有几个优点。(i)它不需要广泛的PCR扩增，也不需要质粒构建通常使用的纯化、修饰和/或连接技术。（ii）该方法不依赖于可用的限制性位点，因此片段和载体DNA可以在DNA序列内连接。这使得在哺乳动物细胞、其他酵母物种、大肠杆菌和其他表达系统中使用多功能克隆载体进行蛋白质表达成为可能。（三）最后，该技术利用廉价和容易获得的酵母菌株、质粒和微生物技术。
We describe a genera method for plasmid assembly that uses yeast and extends beyond yeast-specific research applications. This technology exploits the homologous recombination, double-stranded break repair pathway in Saccharomyces cerevisiae to join DNA fragments. Synthetic, double-stranded "recombination linkers" were used to "subclone" a DNA fragment into a plasmid with >80% efficiency. Quantitative data on the influence of DNA concentration and overlap length on the efficiency of recombination are presented. Using a simple procedure, plasmids were shuttled from yeast into E. coli for subsequent screening and large-scale plasmid preps. This simple method for plasmid construction has several advantages. (i) It bypasses the need for extensive PCR amplification and for purification, modification and/or ligation techniques routinely used for plasmid constructions. (ii) The method does not rely on available restriction sites, thus fragment and vector DNA can be joined within an DNA sequence. This enables the use of multifunctional cloning vectors for protein expression in mammalian cells, other yeast species, E. coli and other expression systems as discussed. (iii) Finally, the technology exploits yeast strains, plasmids and microbial techniques that are inexpensive and readily available.