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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.
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).
Mutations in Microorganisms01:18

Mutations in Microorganisms

Mutations are heritable changes in an organism’s genome involving alterations in the base sequence of DNA or RNA. These changes can influence cellular processes and phenotypic traits, potentially transforming the unaltered wild type into a mutant form. Such changes, termed forward mutations, are pivotal in shaping the genetic diversity of organisms.RNA viruses exhibit the highest mutation rates due to the absence of robust proofreading mechanisms during genome replication. In contrast,...
Mismatch Repair01:20

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
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...

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Related Experiment Video

Updated: May 15, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
09:01

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

Published on: March 16, 2011

PFunkel: efficient, expansive, user-defined mutagenesis.

Elad Firnberg1, Marc Ostermeier

  • 1Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA.

Plos One
|January 4, 2013
PubMed
Summary
This summary is machine-generated.

PFunkel enables efficient, user-defined DNA mutagenesis and library construction in a single day. This method achieves high accuracy for site-directed mutagenesis and comprehensive codon libraries, aiding in the discovery of adaptive mutations.

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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

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Last Updated: May 15, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
09:01

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

Published on: March 16, 2011

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

Published on: May 15, 2018

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • DNA mutagenesis is crucial for understanding gene function and protein engineering.
  • Existing methods can be time-consuming, labor-intensive, and lack flexibility for library construction.

Purpose of the Study:

  • To introduce PFunkel, a novel, versatile method for DNA mutagenesis.
  • To enable efficient construction of researcher-defined DNA libraries.
  • To identify novel adaptive mutations conferring resistance to ß-lactamase inhibitors.

Main Methods:

  • PFunkel utilizes single-stranded (ssDNA) or double-stranded (dsDNA) templates for mutagenesis.
  • The entire process is completed in a single day, in a single tube, without intermediate purification or sub-cloning.
  • Demonstrated site-saturation mutagenesis at four distal sites and comprehensive codon mutagenesis of the TEM-1 ß-lactamase gene.

Main Results:

  • PFunkel achieves site-directed mutagenesis efficiency approaching 100%.
  • A site-saturation library at four distal sites was created with 70% efficiency.
  • A comprehensive codon mutagenesis library of TEM-1 ß-lactamase (18,081 members) showed ~97% of designed substitutions.
  • Identified 18 novel adaptive mutations conferring tazobactam resistance, with three mutations showing high resistance.

Conclusions:

  • PFunkel offers an efficient and versatile platform for DNA mutagenesis and library generation.
  • The method facilitates the discovery of clinically relevant adaptive mutations.
  • PFunkel has significant potential for protein engineering and drug resistance research.