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Related Concept Videos

Exon Recombination02:32

Exon Recombination

The evolution of new genes is critical for speciation. Exon recombination, also known as exon shuffling or domain shuffling, is an important means of new gene formation. It is observed across vertebrates, invertebrates, and in some plants such as potatoes and sunflowers. During exon recombination, exons from the same or different genes recombine and produce new exon-intron combinations, which might evolve into new genes. 
Exon shuffling follows “splice frame rules.” Each exon has three reading...
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...
Base Excision Repair01:54

Base Excision Repair

One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...
Base Excision Repair01:54

Base Excision Repair

One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...
Next-generation Sequencing03:00

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
Nucleotide Excision Repair01:08

Nucleotide Excision Repair

Overview

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Related Experiment Video

Updated: Jun 7, 2026

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
09:16

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Published on: March 25, 2020

Nucleotide exchange and excision technology DNA shuffling and directed evolution.

Janina Speck1, Sabine C Stebel, Katja M Arndt

  • 1Centre for Biological Signalling Studies (bioss), Institute for Biology III, Freiburg, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|October 23, 2010
PubMed
Summary

This study introduces a novel DNA shuffling technique called nucleotide exchange and excision technology (NExT). This method offers a robust and controllable way to create diverse gene libraries for directed evolution.

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Molecular Evolution of the Tre Recombinase
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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetic Engineering

Background:

  • Directed evolution optimizes DNA and protein properties effectively.
  • Existing DNA shuffling methods are limited and difficult to adapt.
  • A need exists for more robust and controllable DNA shuffling techniques.

Purpose of the Study:

  • To develop and demonstrate a novel, efficient, and controllable DNA shuffling method.
  • To overcome limitations of current DNA shuffling protocols.
  • To facilitate the creation of diverse gene libraries for protein engineering.

Main Methods:

  • Nucleotide Exchange and Excision Technology (NExT) for DNA fragmentation.
  • Enzymatic removal of incorporated 'exchange nucleotides' (e.g., uridine).
  • Chemical cleavage, primer extension, and PCR amplification for gene library reassembly.

Main Results:

  • Demonstrated NExT DNA shuffling using chloramphenicol acetyltransferase variants.
  • Achieved an average parental fragment size of 86 bases with 33% dUTP substitution.
  • Observed a low mutation rate of only 0.1% in shuffled clones.
  • Developed NExTProg software to predict fragment size distribution.

Conclusions:

  • NExT provides a robust, efficient, and controllable DNA fragmentation step for DNA shuffling.
  • The method enables the generation of diverse gene libraries with precise control over fragmentation.
  • NExT is a valuable tool for directed evolution and protein engineering.