Linker-mediated recombinational subcloning of large DNA fragments using yeast
Linker-mediated recombinational subcloning of large DNA fragments using yeast
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DOI：
10.1101/gr.205201
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发表时间：
2002-01-01
期刊：
影响因子：
7
通讯作者：
Olson, MV
中科院分区：
文献类型：
--
作者：
Raymond, CK;Sims, EH;Olson, MV
酵母中的同源重组途径是质粒序列特异性组装的理想工具。发现与载体和靶片段重叠的互补的80个核苷酸的寡核苷酸充当片段亚克隆的有效重组接头。使用电穿孔，单链80聚体足以用于常规质粒构建。引入基于环己酰亚胺的反选择以增加通过相对于非特异性载体背景的同源重组克隆的特异性。重建实验表明这种反选择使克隆特异性增加了100倍。环己酰亚胺反选择与80-bp连接子一起用于从细菌人工染色体亚克隆靶向区域。这项技术可能会在完成人类基因组测序计划的最后阶段以及BAC克隆在复杂基因组功能分析中的应用中找到广泛的应用。
The homologous recombination pathway in yeast is an ideal tool for the sequence-specific assembly of plasmids. Complementary 80-nucleotide oligonucleotides that overlap a vector and a target fragment were found to serve as efficient recombination linkers for fragment subcloning. Using electroporation, single-stranded 80-mers were adequate for routine plasmid construction. A cycloheximide-based counterselection was introduced to increase the specificity of cloning by homologous recombination relative to nonspecific vector background. Reconstruction experiments suggest this counterselection increased cloning specificity by 100-fold. Cycloheximide counterselection was used in conjunction with 80-bp linkers to subclone targeted regions from bacterial artificial chromosomes. This technology may find broad application in the Final stages of completing the Human Genome Sequencing Project and in applications of BAC clones to the functional analysis of complex genomes.