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

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

DOI:
10.3791/60807-v

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09:16 min

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March 25, 2020

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

Chapters

  • 00:05Introduction
  • 00:49Split-and-Mix Synthesis of Mutagenic Primers
  • 02:34Performing Compartmentalized In Vitro Transcription-translation Reaction
  • 05:07Continued Processing of Libraries and Selection
  • 07:20Results: Examples of Screening of NlaIV Variants for Sequence Specificity Alteration
  • 08:16Conclusion

Summary

Automatic Translation

Automatic Translation

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.

Tags

In Vitro Directed EvolutionRestriction EndonucleaseSequence SpecificityIn Vitro CompartmentalizationDirected EvolutionSubsaturation MutagenesisOligonucleotide SynthesisOil Surfactant MixtureLibrary Preparation

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