Engineering novel restriction endonucleases: principles and applications
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Updated: Nov 4, 2026

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
1Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Giessen, Germany. albert.jeltsch@chemie.bio.uni-giessen.de
Abstract:
Restriction endonucleases cleave DNA with remarkable sequence specificity. In this review, we summarize the status of, and prospects for, engineering restriction endonucleases with new specificities. Such variants could be of considerable commercial value because restriction enzymes are among the most frequently used enzymes in molecular biology, and not all the desirable specificities are available. While it has not yet been possible to effect specificity changes, mutant have been described that (1) exhibit relaxed specificity, (2) favour modified substrates over their natural substrates, (3) discriminate between cleavage sites located in different sequences, (4) prefer metal ions other than Mg2+ as cofactors for cleavage, or (5) possess site-specific DNA-nicking activity.
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