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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.
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.
PCR01:32

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Related Experiment Video

Updated: Jul 15, 2026

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast
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Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Published on: May 15, 2018

Error-prone rolling circle amplification: the simplest random mutagenesis protocol.

Ryota Fujii1, Motomitsu Kitaoka, Kiyoshi Hayashi

  • 1National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki, 305-8642, Japan.

Nature Protocols
|April 5, 2007
PubMed
Summary

Error-prone rolling circle amplification (RCA) offers a simple method for creating mutant libraries. This technique rapidly generates plasmids with random point mutations, facilitating wider adoption of random mutagenesis.

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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast
07:18

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Published on: March 16, 2011

Area of Science:

  • Molecular Biology
  • Biotechnology

Background:

  • Random mutagenesis is crucial for genetic studies and protein engineering.
  • Existing methods for random mutagenesis can be complex and time-consuming.

Purpose of the Study:

  • To describe a simple and rapid protocol for generating randomly mutated plasmid libraries.
  • To enable wider adoption of random mutagenesis techniques.

Main Methods:

  • Error-prone rolling circle amplification (RCA) using a template plasmid and MnCl2.
  • Amplification followed by host strain transformation to create a mutant library.
  • Protocol requires minimal optimization, with a short reaction preparation time.

Main Results:

  • Generated mutant libraries with three to four random point mutations per kilobase.
  • Achieved random mutagenesis throughout the entire plasmid.
  • Demonstrated the simplicity and speed of the error-prone RCA protocol.

Conclusions:

  • Error-prone RCA is a straightforward and efficient method for creating diverse mutant plasmid libraries.
  • The protocol's simplicity reduces the need for specialized reagents or conditions.
  • This method has the potential to significantly increase the accessibility of random mutagenesis.