SITE-SPECIFIC DELETION OF THE N-TERMINAL SEGMENTS FROM EcoRI ENDONUCLEASE USING THE POLYMERASE CHAIN REACTION  

作　　者：杨香娇 陈常庆 王德宝 杨胜利 

机构地区：[1]Shanghai Institute of Biochemistry, Academia Sinica, Shanghai 200031, PRC [2]Shanghai Center of Biotechnology, Academia Sinica, Shanghai 200031, PRC

出　　处：《Science China Chemistry》1991年第12期1436-1443,共8页中国科学（化学英文版）

摘　　要：We developed a general and efficient method for directing the deletions of DNA sequences of any lengths using polymerase chain reaction (PCR). The method was based on in vitro amplification of target sequences with site-specific deletions, Klenow end-flushing and blunt-end cloning. As an example, it was used to delete the restriction gene encoding EcoRI endonuclease, resulting in plasmids expressing two truncated forms. Assays using SDS-PAGE and gel retardation revealed the important role of the amphipathic helix (29-43) of the EcoRI endonuclease in binding to its cognate substrate.We developed a general and efficient method for directing the deletions of DNA sequences of any lengths using polymerase chain reaction (PCR). The method was based on in vitro amplification of target sequences with site-specific deletions, Klenow end-flushing and blunt-end cloning. As an example, it was used to delete the restriction gene encoding EcoRI endonuclease, resulting in plasmids expressing two truncated forms. Assays using SDS-PAGE and gel retardation revealed the important role of the amphipathic helix (29-43) of the EcoRI endonuclease in binding to its cognate substrate.

关 键 词：PCR SITE-SPECIFIC DELETION ECORI endonuclease. 

分 类 号：O6[理学—化学]