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In-vitro Mutagenesis

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: Sep 7, 2025

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

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

Published on: May 15, 2018

Rapid Cloning of Random Mutagenesis Libraries Using PTO-QuickStep.

Pawel Jajesniak¹, Kang Lan Tee¹, Tuck Seng Wong²

¹Department of Chemical and Biological Engineering, ChELSI Institute and Advanced Biomanufacturing Centre, University of Sheffield, Sheffield, England, UK.

Methods in Molecular Biology (Clifton, N.J.)

|June 21, 2022

Summary

PTO-QuickStep is a novel molecular cloning method for seamless DNA integration into plasmids. This efficient technique accelerates protein engineering and synthetic biology applications without complex purification or enzymatic steps.

Keywords:

DNA assembly Directed evolution Metabolic engineering Molecular cloning Protein engineering Random mutagenesis Recombinant DNA Synthetic biology

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Last Updated: Sep 7, 2025

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Published on: May 15, 2018

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Published on: July 3, 2016

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

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Published on: April 1, 2016

Area of Science:

Molecular Biology
Biotechnology
Synthetic Biology

Background:

Traditional molecular cloning methods can be time-consuming and labor-intensive.
Existing techniques often require multiple steps, including DNA gel purification and enzymatic restriction and ligation.
There is a need for faster, more efficient cloning strategies to accelerate research in protein engineering and synthetic biology.

Purpose of the Study:

To introduce PTO-QuickStep, a novel, rapid, and cost-effective molecular cloning technique.
To demonstrate the versatility of PTO-QuickStep for seamless DNA fragment integration into plasmids.
To highlight the utility of PTO-QuickStep in accelerating protein engineering and synthetic biology workflows.

Main Methods:

PTO-QuickStep enables seamless point integration of DNA fragments into any plasmid position.
The method eliminates the need for DNA gel purification and enzymatic restriction and ligation steps.
It allows for the integration of random mutagenesis (e.g., error-prone PCR) within the cloning workflow.

Main Results:

PTO-QuickStep provides a time-efficient and cost-effective molecular cloning solution.
The technique facilitates rapid cloning of random mutagenesis libraries for directed evolution.
Noncoding DNA fragments can be quickly integrated to modify existing plasmids.

Conclusions:

PTO-QuickStep is a versatile and efficient tool for molecular cloning, protein engineering, and synthetic biology.
The method significantly reduces the time and cost associated with plasmid modification.
PTO-QuickStep accelerates research by simplifying and speeding up DNA integration and library construction.