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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.
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...
Spontaneous and Induced Mutations01:30

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).

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Related Experiment Video

Updated: May 11, 2026

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
11:36

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Published on: July 3, 2016

Library construction and evaluation for site saturation mutagenesis.

Bradford Sullivan1, Adam Z Walton, Jon D Stewart

  • 1Department of Chemistry, University of Florida, 126 Sisler Hall, Gainesville, FL 32611, USA.

Enzyme and Microbial Technology
|May 21, 2013
PubMed
Summary

We created a method for site-saturation libraries, achieving 27.4±3.0 codons per 32 possible. This technique, validated with alkene reductase OYE 2.6, includes quality analysis for efficient library selection.

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

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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

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Last Updated: May 11, 2026

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing
11:36

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Published on: July 3, 2016

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

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A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates
11:49

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Published on: August 21, 2018

Area of Science:

  • Biotechnology
  • Molecular Biology
  • Enzyme Engineering

Background:

  • Site-saturation mutagenesis is crucial for protein engineering.
  • Developing efficient methods for creating and evaluating these libraries is essential for identifying improved protein variants.

Purpose of the Study:

  • To develop and validate a robust method for generating and assessing site-saturation libraries.
  • To introduce a quantitative analysis for predicting library quality early in the construction process.

Main Methods:

  • Optimized PCR primer design and transformation efficiency protocols.
  • Creation and sequencing of 11 independent site-saturation libraries for alkene reductase OYE 2.6.
  • Development of a quantitative analysis (Q-values) based on fluorescence sequencing data to assess library degeneracy.

Main Results:

  • Consistently achieved an average of 27.4±3.0 codons out of 32 possible within 95 transformants.
  • Sequencing of 95 members from 11 libraries confirmed the method's reproducibility.
  • Predicted library degeneracy from early-stage pooled plasmids closely matched observed degeneracy after sequencing ~1000 members.

Conclusions:

  • The developed method provides a reliable way to create high-quality site-saturation libraries.
  • The Q-value analysis enables early identification and rejection of substandard libraries, saving resources.
  • This approach is particularly beneficial for screening methods requiring lower throughput or targeting multiple codon randomization.