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Related Concept Videos

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.
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...
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: Jun 12, 2026

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

A new method for multi-site-directed mutagenesis.

Jing Tian1, Qiong Liu, Sheng Dong

  • 1Shenzhen Uinversity, People's Republic of China.

Analytical Biochemistry
|June 16, 2010
PubMed
Summary

Researchers developed a simplified method for multi-site-directed mutagenesis using polymerase chain reaction (PCR) and DpnI digestion. This cost-effective technique enables efficient genetic modification, even for closely spaced mutations.

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Related Experiment Videos

Last Updated: Jun 12, 2026

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

Homemade Site Directed Mutagenesis of Whole Plasmids
07:11

Homemade Site Directed Mutagenesis of Whole Plasmids

Published on: May 11, 2009

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

Area of Science:

  • Molecular Biology
  • Genetic Engineering
  • Biotechnology

Background:

  • Multi-site-directed mutagenesis is crucial for studying gene function and protein engineering.
  • Existing methods can be complex, costly, and time-consuming, especially for introducing multiple mutations.
  • The need for simplified, efficient, and economical mutagenesis techniques is significant in molecular biology research.

Purpose of the Study:

  • To develop a modified, streamlined method for multi-site-directed mutagenesis.
  • To enable efficient introduction of multiple mutations, including those in close proximity.
  • To reduce the complexity and cost associated with standard mutagenesis protocols.

Main Methods:

  • The method utilizes polymerase chain reaction (PCR), DpnI digestion, and overlap extension PCR.
  • It requires only methylated plasmids, obtainable via Dam methyltransferase or from dam(+) Escherichia coli.
  • The procedure bypasses the need for 5'-phosphorylated primers and ligation steps.

Main Results:

  • A robust and simplified protocol for multi-site-directed mutagenesis was successfully established.
  • The method demonstrated efficacy in introducing multiple mutations, even at close proximity.
  • Significant reduction in experimental complexity and cost compared to traditional methods was achieved.

Conclusions:

  • The developed modified method offers a simplified and cost-effective approach for multi-site-directed mutagenesis.
  • This technique is suitable for researchers needing to introduce multiple genetic modifications efficiently.
  • The protocol's ease of use and reduced cost can accelerate research in molecular biology and genetic engineering.