摘要
重组工程(recombineering)是近几年来兴起的一种基于体内同源重组的、新型的遗传工程技术。作为重组工程应用方式之一的空隙修复(gap-repair),是一种捕捉和克隆目的DNA的方法,具有操作简单、步骤少,没有突变、保真度高,不受酶切位点限制等等优点。以pACYC184为模板,PCR扩增含p15A复制子、氯霉素抗性基因和对S.cerevisiaeALD4基因同源臂的线性片段,与酵母染色体DNA共同电击转化诱导型表达了λ噬菌体重组酶活性的大肠杆菌BW25113(pKD46)感受态细胞,通过空隙修复方式,成功地从酵母染色体DNA直接捕捉到大小为1 016bp的ALD4基因部分区段,得到3188bp的重组质粒pACYC184-ALD4。为进一步掌握和充分利用该技术直接捕捉更大片段基因打下了基础。
Based on principle of homologous recombination, a new genetic engineering system named ＂recombineering＂ has been developed in recent years. Gap-repair is an applicable technology of the recombineering, especially useful in capturing and cloning the target DNA fragments in vivo. It holds a lot of advantages including simple and rapid operation,high-fidelity against mutation,free of restriction enzymes participating in the reaction. A linear PCR fragment containing p15A replication origin,Cm selective marker and homologous regions from S.cerevisiae ALD4 gene was amplified from pACYC184 vector. The linear PCR fragment was mixed with S.cerevisiae genomic DNA and introduced into inducible BW25113 （pKD46）competent cells by means of co-electroporation. In this homologous recombination system,the 1 016bp ALD4 DNA of S.cerevisiae was transferred into PCR fragment directly and a 3188bp pACYC184-ALD4 recombinant plasmid was produced by gap-repair. This result would be helpful to learn and make use of the gap-repair technology in capturing larger DNA fragments.
出处
《生物技术通报》
CAS
CSCD
2007年第2期124-127,150,共5页
Biotechnology Bulletin
