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Restriction Enzymes

Restriction Enzymes

Restriction enzymes are bacterial enzymes used to cut DNA in a sequence-specific manner. To cleave DNA, they bind to specific palindromic sequences called restriction sites. Such palindromic DNA sequences or inverted repeats are commonly found in regions of functional significance, such as the origin of replication, gene operator sites, and regions containing transcription termination signals.
The host bacteria protect their own genomic DNA from these enzymes by methylating these sites. Some...

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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...

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Updated: Mar 7, 2026

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

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

Published on: March 25, 2020

Programmable DNA-Guided Artificial Restriction Enzymes.

Behnam Enghiad¹, Huimin Zhao¹

¹Department of Chemical and Biomolecular Engineering, ‡Carl R. Woese Institute for Genomic Biology, Department of Chemistry, Department of Biochemistry, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.

ACS Synthetic Biology

|February 7, 2017

Summary

This summary is machine-generated.

Researchers developed artificial restriction enzymes (AREs) using Pyrococcus furiosus Argonaute (PfAgo). These novel enzymes offer programmable DNA cleavage at any site, advancing molecular biology tools.

Keywords:

DNA cloning DNA profiling recombinant DNA technology restriction enzymes

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Area of Science:

Molecular Biology
Biotechnology
Enzymology

Background:

Restriction enzymes are crucial for recombinant DNA technology but have limitations in specificity and availability.
Existing tools hinder precise DNA manipulation for advanced biological research.

Purpose of the Study:

To engineer artificial restriction enzymes (AREs) using a Pyrococcus furiosus Argonaute (PfAgo) platform.
To overcome the limitations of traditional restriction enzymes by enabling targeted DNA cleavage at arbitrary sites.

Main Methods:

Utilized a PfAgo-based system guided by short DNA sequences for targeted DNA cleavage.
Operated the system at high temperatures (>87 °C) followed by single-stranded DNA reannealing.
Generated and validated over 18 AREs for applications like DNA fingerprinting and molecular cloning.

Main Results:

Successfully generated AREs capable of recognizing and cleaving DNA at virtually any arbitrary site.
Achieved defined sticky ends of varying lengths post-cleavage.
Demonstrated AREs function with high efficiency, comparable to or exceeding natural counterparts.

Conclusions:

The PfAgo-based platform provides a highly programmable, versatile, and efficient method for creating novel restriction enzymes.
This technology significantly expands the toolkit for DNA manipulation, molecular cloning, and DNA fingerprinting.
AREs represent a significant advancement over traditional restriction enzymes, offering unprecedented control in genetic engineering.