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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.
The recognition sites for Cre recombinase called LoxP...
RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the...
Ribozymes02:47

Ribozymes

The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can be...
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...

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Related Experiment Video

Updated: May 17, 2026

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
09:16

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

Published on: March 25, 2020

Engineering RNA endonucleases with customized sequence specificities.

Rajarshi Choudhury1, Yihsuan S Tsai, Daniel Dominguez

  • 1Department of Pharmacology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

Nature Communications
|October 25, 2012
PubMed
Summary
This summary is machine-generated.

Researchers engineered artificial RNA endonucleases for precise RNA targeting. These novel enzymes enable specific RNA cleavage for in vitro manipulation and in vivo gene silencing applications.

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Last Updated: May 17, 2026

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
09:16

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Published on: March 25, 2020

Substrate Generation for Endonucleases of CRISPR/Cas Systems
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Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes
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Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

Published on: July 5, 2024

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Synthetic Biology

Background:

  • Specific RNA cleavage is essential for molecular biology techniques and gene regulation.
  • Existing methods for RNA manipulation and gene silencing have limitations in specificity and versatility.

Purpose of the Study:

  • To engineer artificial site-specific RNA endonucleases analogous to DNA restriction enzymes.
  • To develop a versatile tool for targeted RNA manipulation and gene silencing.

Main Methods:

  • Combined a general RNA cleavage domain with Pumilio/fem-3-binding factor domains.
  • Designed artificial endonucleases to recognize specific 8-nucleotide RNA sequences.
  • Tested cleavage efficiency and specificity on various RNA substrates.

Main Results:

  • Successfully engineered artificial site-specific RNA endonucleases with designed RNA-binding specificities.
  • Demonstrated efficient and specific cleavage of target RNA sequences in vitro.
  • Showcased gene silencing capabilities in bacterial (Escherichia coli) and human cells.

Conclusions:

  • Artificial site-specific RNA endonucleases are effective tools for precise RNA manipulation in vitro.
  • These engineered nucleases offer a promising platform for sequence-specific gene silencing in diverse biological systems.