Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

6.0K
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...
6.0K
Recombinant DNA01:09

Recombinant DNA

93.9K
Overview
93.9K
Homologous Recombination02:31

Homologous Recombination

50.4K
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
50.4K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Engineering the probiotic <i>Escherichia coli</i> Nissle 1917 as a host organism for coliphage production.

Synthetic and systems biotechnology·2026
Same author

Orchestrating triacylglycerol biosynthesis and hydrolysis via synthetic genetic circuits enhances spinosad production in Streptomyces albus.

Bioresource technology·2026
Same author

Endoplasmic Reticulum-Targeted Biomimetic Nanoparticles Potentiate the Immunotherapy of Triple-Negative Breast Cancer by Improving Immunogenicity and Eliminating Immune Resistance.

ACS nano·2026
Same author

Development of an efficient genome editing system using recombinase-mediated cassette exchange in <i>E. coli</i> Nissle 1917 for large gene cluster integration and heterologous astaxanthin production.

Synthetic and systems biotechnology·2026
Same author

Unraveling the Charge Rearrangement-Driven Synergistic Mechanism in SiO<sub>2</sub>@Ni-Co/CNTs: The Regulatory Role of Ni-Doping in the Bimetallic Shell.

ACS applied materials & interfaces·2026
Same author

High Mobility Group Protein B1 Promotes Interferon Regulatory Factor 1 SUMOylation to Prime Trained Immunity of Circulating Monocytes and Aggravate the Progressive Synovial Inflammation in Knee Osteoarthritis.

Research (Washington, D.C.)·2026

Related Experiment Video

Updated: Jun 21, 2025

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
07:04

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Published on: February 5, 2019

19.9K

Seamless site-directed mutagenesis in complex cloned DNA sequences using the RedEx method.

Ji Luan1, Chaoyi Song1, Yan Liu1

  • 1State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Helmholtz International Lab for Anti-infectives, Shandong University-Helmholtz Institute of Biotechnology, Shandong University, Qingdao, Shandong, China.

Nature Protocols
|July 15, 2024
PubMed
Summary
This summary is machine-generated.

The RedEx strategy enables accurate seamless site-directed mutagenesis in large DNA molecules like bacterial artificial chromosomes (BACs), even with repetitive sequences. This method simplifies genetic element modification for researchers.

More Related Videos

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

7.3K
Homemade Site Directed Mutagenesis of Whole Plasmids
07:11

Homemade Site Directed Mutagenesis of Whole Plasmids

Published on: May 11, 2009

33.2K

Related Experiment Videos

Last Updated: Jun 21, 2025

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli
07:04

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Published on: February 5, 2019

19.9K
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

7.3K
Homemade Site Directed Mutagenesis of Whole Plasmids
07:11

Homemade Site Directed Mutagenesis of Whole Plasmids

Published on: May 11, 2009

33.2K

Area of Science:

  • Molecular Biology
  • Genetic Engineering
  • Biotechnology

Background:

  • Seamless site-directed mutagenesis is crucial for protein function studies and genetic modification.
  • Modifying large DNA molecules, especially those with repetitive sequences like bacterial artificial chromosomes (BACs), presents significant challenges.
  • Existing methods often struggle with accuracy due to unwanted recombination events.

Purpose of the Study:

  • To present the RedEx strategy for efficient and accurate seamless site-directed mutagenesis.
  • To demonstrate the application of RedEx in modifying large plasmids and BACs containing repetitive sequences.
  • To provide a detailed protocol for researchers to implement this technique.

Main Methods:

  • Developed the RedEx strategy for seamless DNA insertion, deletion, and substitution.
  • Utilized recombineering to insert a RedEx cassette with desired mutations and markers.
  • Employed restriction digestion to remove markers and exonuclease-mediated annealing for BAC circularization.

Main Results:

  • Achieved over 80% accuracy in seamless DNA manipulation within large, repetitive gene clusters.
  • Successfully circumvented unwanted recombination events common in previous mutagenesis methods.
  • The protocol is adaptable for large multimodular polyketide synthase gene clusters.

Conclusions:

  • The RedEx strategy offers a highly accurate and efficient method for seamless site-directed mutagenesis in challenging DNA constructs.
  • This technique accelerates research by simplifying the modification of large plasmids and BACs.
  • The detailed protocol enables researchers with DNA cloning experience to master this advanced mutagenesis approach.