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A METHOD FOR ENGINEERING STRAND-SPECIFIC, SEQUENCE-SPECIFIC, DNA-NICKING ENZYMES
A METHOD FOR ENGINEERING STRAND-SPECIFIC, SEQUENCE-SPECIFIC, DNA-NICKING ENZYMES
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机译:一种工程特定序列,特定序列,DNA克隆酶的方法
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摘要
Methods are provided for converting into a sequence specific strand specific and location specific DNA nicking endonuclease, a restriction endonuclease that recognizes an asymmetric DNA sequence, the endonuclease having two catalytic sites and one or more single sequence specific DNA-binding domains. In one embodiment the method requires inactivating one of the catalytic sites of the restriction endonuclease. In another embodiment, the restriction endonuclease is a dimer having a first and second subunit each comprising a sequence specific DNA binding domain, a catalytic site and a dimerization domain. The nicking endonuclease is formed from combining one subunit having an inactivated catalytic site and a second subunit having an inactivated DNA binding domain. The nicking endonuclease may be converted into a restriction endonuclease by the addition of manganese cations in the digestion buffer.
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