Szent-Gyorgyi C
Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.
Yeast. 1996 Jun 15;12(7):667-72. doi: 10.1002/(SICI)1097-0061(19960615)12:7%3C667::AID-YEA955%3E3.0.CO;2-A.
A strategy for gene replacement in Saccharomyces cerevisiae has been modified to facilitate the repeated substitution of a chromosomal locus with in vitro generated variant sequences, so that the resulting locus contains only the desired mutation and is free of extraneous vector DNA. The construction of an internally deleted chromosomal target locus carrying the counterselectable CYH2S marker and a second positively selectable marker has been simplified; the design of the locus has been altered to increase the frequency of authentic gene replacements obtained upon the subsequent integration of in vitro mutated DNA. The modified chromosomal target locus is amenable to replacement using either of two transformation protocols: (i) integration of a second positively selectable plasmid carrying mutant sequences to form a tandem intermediate structure at the locus; upon counterselection on cycloheximide, all vector sequence is excised to give the desired replacement at high frequency (> 70%); (ii) single-step integration of a linear segment of mutated genomic DNA by selection for cycloheximide resistance. A subsequent screen for the loss of the positively selectable target locus marker detects the desired replacement at modest frequency (> 2%). Polymerase chain reaction using multiple primers in a single amplification reaction is useful for monitoring these variously modified chromosomal loci.
一种用于酿酒酵母基因替换的策略已被改进，以促进用体外产生的变异序列对染色体位点进行重复替换，从而使所得位点仅包含所需突变且不含无关载体DNA。携带反选择CYH2S标记和第二个正选择标记的内部缺失染色体靶位点的构建已得到简化；该位点的设计已改变，以提高在随后整合体外突变DNA时获得真实基因替换的频率。修饰后的染色体靶位点适合使用两种转化方案中的任何一种进行替换：（i）整合携带突变序列的第二个正选择质粒，在该位点形成串联中间结构；在对环己酰亚胺进行反选择时，所有载体序列被切除，以高频率（>70%）产生所需替换；（ii）通过选择对环己酰亚胺的抗性，单步整合突变基因组DNA的线性片段。随后对正选择靶位点标记缺失的筛选以适度频率（>2%）检测到所需替换。在单个扩增反应中使用多个引物的聚合酶链反应可用于监测这些经过各种修饰的染色体位点。
