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JoVE Journal
Genetics

This content is Open Access.

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
 

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Article doi: 10.3791/60807
March 25th, 2020

Summary March 25th, 2020

Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.

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