| SITE-SPECIFIC DELETION OF THE N-TERMINAL SEGMENTS FROM EcoRI ENDONUCLEASE USING THE POLYMERASE CHAIN REACTION |
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| 作者姓名： | 杨香娇 陈常庆 王德宝 杨胜利 |
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| 作者单位： | Shanghai Institute of Biochemistry，Academia Sinica，Shanghai 200031，PRC，Shanghai Institute of Biochemistry，Academia Sinica，Shanghai 200031，PRC，Shanghai Institute of Biochemistry，Academia Sinica，Shanghai 200031，PRC，Shanghai Center of Biotechnology，Academia Sinica，Shanghai 200031，PRC |
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| 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.
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SITE-SPECIFIC DELETION OF THE N-TERMINAL SEGMENTS FROM EcoRI ENDONUCLEASE USING THE POLYMERASE CHAIN REACTION |
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| YANG XIANG-JIAO CHEN CHANG-QING WANG DE-BAO.SITE-SPECIFIC DELETION OF THE N-TERMINAL SEGMENTS FROM EcoRI ENDONUCLEASE USING THE POLYMERASE CHAIN REACTION[J].Science in China(Chemistry),1991(12). |
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| Authors: | YANG XIANG-JIAO CHEN CHANG-QING WANG DE-BAO |
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| Abstract: | 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. |
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| Keywords: | PCR site-specific deletion EcoRI endonuclease |
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