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In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
JoVE Journal
Genetica
This content is Free Access.
JoVE Journal Genetica
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

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

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09:16 min

March 25, 2020

DOI:

09:16 min
March 25, 2020

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Summary

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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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