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

DOI :

10.3791/60807-v

9:16 min

March 25th, 2020

March 25th, 2020

6,774 Views

1International Institute of Molecular and Cell Biology, Warsaw (IIMCB)

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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In Vitro Directed Evolution

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