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Related Concept Videos

Restriction Enzymes01:11

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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Updated: May 12, 2026

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
09:53

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

Published on: February 7, 2021

Massively parallel characterization of restriction endonucleases.

Nick Kamps-Hughes1, Aine Quimby, Zhenyu Zhu

  • 1Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA.

Nucleic Acids Research
|April 23, 2013
PubMed
Summary

New sequencing methods accurately measure restriction endonuclease activity on complex DNA. This reveals how flanking sequences influence enzyme specificity and identifies engineered variants with improved fidelity.

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Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
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Last Updated: May 12, 2026

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage
09:53

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

Published on: February 7, 2021

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)
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High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

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Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
06:51

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Published on: May 6, 2020

Area of Science:

  • Molecular Biology
  • Genomics
  • Enzymology

Background:

  • Restriction endonucleases (REs) are enzymes crucial for DNA manipulation.
  • RE activity is influenced by sequence context, enzyme concentration, and buffer conditions.
  • Traditional assays for RE activity use simple DNA substrates like plasmids or oligonucleotides.

Purpose of the Study:

  • To develop and apply high-throughput sequencing-based strategies for assaying RE sequence specificity and flanking sequence preference.
  • To quantitatively characterize the cognate and star site activity of specific REs on complex genomic DNA.
  • To evaluate the impact of engineered high-fidelity RE variants on star activity and flanking sequence influence.

Main Methods:

  • Utilized fragmented genomic DNA from sequenced genomes as complex substrates for RE cleavage assays.
  • Employed high-throughput Illumina sequencing to quantify RE cleavage across millions of sites in a single reaction.
  • Mapped sequencing reads to Escherichia coli and Drosophila melanogaster genomes to analyze RE activity and specificity.

Main Results:

  • Quantitatively characterized cognate and star site activity for EcoRI and MfeI restriction endonucleases.
  • Demonstrated genome-wide decreases in star activity for engineered high-fidelity variants (EcoRI-HF and MfeI-HF).
  • Quantified the influence of flanking nucleotides on MfeI cleavage preference.

Conclusions:

  • Developed robust, high-throughput sequencing-based methods for detailed analysis of RE activity on complex DNA.
  • Provided quantitative insights into RE sequence specificity, flanking sequence effects, and the improved fidelity of engineered variants.
  • The presented methods are broadly applicable to type II restriction endonucleases for comprehensive activity profiling.