Raymond C K, Pownder T A, Sexson S L
ZymoGenetics, Seattle, WA, USA.
Biotechniques. 1999 Jan;26(1):134-8, 140-1. doi: 10.2144/99261rr02.
We describe a general 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 any 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.
我们描述了一种利用酵母的质粒组装通用方法，该方法超出了酵母特异性研究应用的范围。这项技术利用酿酒酵母中的同源重组双链断裂修复途径来连接DNA片段。合成的双链“重组接头”用于将DNA片段“亚克隆”到质粒中，效率超过80%。文中给出了关于DNA浓度和重叠长度对重组效率影响的定量数据。通过一个简单的程序，质粒从酵母转移到大肠杆菌中，以便后续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）它无需进行广泛的PCR扩增以及常规用于质粒构建的纯化、修饰和/或连接技术。（ii）该方法不依赖于可用的限制性酶切位点，因此片段和载体DNA可以在任何DNA序列内连接。如所讨论的，这使得多功能克隆载体可用于在哺乳动物细胞、其他酵母物种、大肠杆菌和其他表达系统中进行蛋白质表达。（iii）最后，该技术利用的酵母菌株、质粒和微生物技术价格低廉且易于获得。
