PCR-directed in vivo plasmid construction using homologous recombination in baker's yeast.
PCR-directed in vivo plasmid construction using homologous recombination in baker's yeast.
复制标题
使用面包酵母中的同源重组进行 PCR 指导的体内质粒构建。
DOI：
10.1007/978-1-61779-228-1_24
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发表时间：
2011
期刊：
影响因子：
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通讯作者：
Andersen,ErikC
中科院分区：
文献类型：
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作者：
Andersen,ErikC
各种各样的应用需要创建定制设计的质粒，包括转基因报告、异源基因融合和表型抢救质粒。这些质粒传统上是使用限制性内切酶和体外连接反应来创建的，但这些技术依赖于可用的限制位点，并且考虑到要连接的片段的大小和数量，可能会很费力。面包师的酵母酿酒酵母提供了一个强大的平台，通过聚合酶链式反应指导酵母介导的连接来创造几乎任何质粒。这项技术可以在体内连接高达50千碱基对(Kb)的复杂质粒，以产生具有精确定义序列的质粒。
A variety of applications require the creation of custom-designed plasmids, including transgenic reporters, heterologous gene fusions, and phenotypic rescue plasmids. These plasmids are created traditionally using restriction digests and in vitro ligation reactions, but these techniques are dependent on available restriction sites and can be laborious given the size and number of fragments to be ligated. The baker’s yeastSaccharomyces cerevisiaeprovides a powerful platform to create nearly any plasmid through PCR-directed yeast-mediated ligation. This technique can ligate complex plasmids of up to 50 kilobasepairs (kb) in vivo to produce plasmids with precisely defined sequences.