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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...
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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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CRISPR01:59

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short...
CRISPR01:59

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CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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...
In-vitro Mutagenesis01:16

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

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Related Experiment Video

Updated: Jul 18, 2026

Mouse Genome Engineering Using Designer Nucleases
12:04

Mouse Genome Engineering Using Designer Nucleases

Published on: April 2, 2014

Gene manipulation using artificial restriction DNA cutter.

Yoji Yamamoto1, Jing-Min Zhou, Takafumi Tomita

  • 1Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.

Nucleic Acids Symposium Series (2004)
|December 8, 2006
PubMed
Summary

We developed a novel Artificial Restriction DNA Cutter (ARCUT) for efficient DNA manipulation. This tool enables seamless insertion of PCR products into plasmids and facilitates PCR-free chimera protein construction in E. coli.

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Area of Science:

  • Molecular Biology
  • Genetic Engineering
  • Biotechnology

Background:

  • Plasmid DNA manipulation is crucial for genetic engineering.
  • Existing methods for DNA insertion can be complex and time-consuming.
  • The development of novel tools for precise DNA manipulation is an ongoing area of research.

Purpose of the Study:

  • To introduce and validate a newly developed Artificial Restriction DNA Cutter (ARCUT) system.
  • To demonstrate the utility of ARCUT for efficient plasmid DNA manipulation.
  • To showcase ARCUT's application in PCR-free chimera protein construction.

Main Methods:

  • Utilized ARCUT for the insertion of a Polymerase Chain Reaction (PCR) product into the pBR322 plasmid vector.
  • Transfected Escherichia coli (E. coli) cells with the resulting recombinant plasmid DNA.
  • Assessed the integrity of the recombinant DNA and the functionality of the ARCUT system through cell growth and subsequent protein analysis.

Main Results:

  • Successful insertion of PCR product into pBR322 plasmid using ARCUT.
  • E. coli cells transfected with recombinant plasmid DNA exhibited successful growth, indicating successful recombination.
  • Recombination proceeded without detectable unexpected mutations, validating the precision of ARCUT.
  • Demonstrated the application of ARCUT for PCR-free chimera protein construction.

Conclusions:

  • ARCUT is an effective tool for precise manipulation of plasmid DNA.
  • The ARCUT system facilitates efficient and mutation-free insertion of DNA fragments.
  • ARCUT offers a valuable alternative for PCR-free chimera protein construction, streamlining genetic engineering workflows.