重组工程
同源重组
生物
基因组工程
遗传学
基因
基因靶向
计算生物学
体外重组
DNA
基因组
合成生物学
基因组编辑
大肠杆菌
分子克隆
肽序列
作者
Shyam K. Sharan,Lynn C. Thomason,Sergey G. Kuznetsov,Donald L. Court
出处
期刊:Nature Protocols
[Springer Nature]
日期:2009-01-29
卷期号:4 (2): 206-223
被引量:749
标识
DOI:10.1038/nprot.2008.227
摘要
Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in Escherichia coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-stranded linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted and regions of bacterial artificial chromosomes or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about 1 week's time.
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