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Spontaneous and Induced Mutations

Spontaneous and Induced Mutations

Spontaneous mutations arise infrequently during DNA replication due to errors in the process. A key factor behind these errors is tautomeric shifts in nitrogenous bases, where bases transition from keto to enol forms or amino to imino forms. This shift can alter base-pairing rules, leading to mutations. Additionally, reactive oxygen species (ROS) arising from aerobic metabolism can damage DNA, resulting in depurination (loss of a purine base) or depyrimidination (loss of a pyrimidine base).

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.

Conservative Site-specific Recombination and Phase Variation

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...

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Updated: Sep 29, 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

Fragment-Directed Random Mutagenesis by the Reverse Kunkel Method.

Wen-Ching Lin¹, Hao-Cheng Tang¹, Han Ying Wang¹

¹Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan.

ACS Synthetic Biology

|March 24, 2022

Summary

This summary is machine-generated.

We developed the reverse Kunkel method, a novel protein engineering technique that combines user-defined and random mutagenesis. This method efficiently generates improved antibodies and is ideal for engineering complex proteins like Cas9.

Keywords:

Kunkel method affinity maturation phage display protein engineering random mutagenesis site-directed mutagenesis yeast surface display

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Area of Science:

Protein Engineering
Molecular Biology
Biotechnology

Background:

Traditional protein engineering relies on user-defined or random mutagenesis, each with limitations.
A need exists for methods combining targeted control with random mutation introduction.

Purpose of the Study:

To introduce a novel mutagenesis protocol, the reverse Kunkel method.
To combine the advantages of user-defined and random mutagenesis in a single reaction.
To engineer improved proteins, particularly antibodies.

Main Methods:

Developed the reverse Kunkel method for one-pot, multi-region random mutagenesis.
Mimicked somatic hypermutation in antibodies, focusing mutations on complementarity-determining regions.
Utilized phage display and yeast display for selection of improved variants.

Main Results:

Successfully generated antibodies with significantly enhanced affinity and immunostaining performance.
Demonstrated the method's efficacy against a model protein and a neurotransmitter peptide.
Validated the reverse Kunkel method's applicability for complex protein engineering.

Conclusions:

The reverse Kunkel method offers a powerful approach for protein engineering.
It is particularly suitable for proteins with multiple variable regions or discontinuous functional domains.
This technique advances the engineering of antibodies, adeno-associated virus capsids, and enzymes like Cas9 and Cas12a.