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Related Concept Videos

Gene Conversion02:08

Gene Conversion

Other than maintaining genome stability via DNA repair, homologous recombination plays an important role in diversifying the genome. In fact, the recombination of sequences forms the molecular basis of genomic evolution. Random and non-random permutations of genomic sequences create a library of new amalgamated sequences. These newly formed genomes can determine the fitness and survival of cells. In bacteria, homologous and non-homologous types of recombination lead to the evolution of new...
Fixing Double-strand Breaks02:04

Fixing Double-strand Breaks

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
Fixing Double-strand Breaks02:04

Fixing Double-strand Breaks

The double-stranded structure of DNA has two major advantages. First, it serves as a safe repository of genetic information where one strand serves as the back-up in case the other strand is damaged. Second, the double-helical structure can be wrapped around proteins called histones to form nucleosomes, which can then be tightly wound to form chromosomes. This way, DNA chains up to 2 inches long can be contained within microscopic structures in a cell. A double-stranded break not only damages...
Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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...
Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart, a...

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Related Experiment Video

Updated: May 31, 2026

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
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Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Published on: June 8, 2018

Tracking genome engineering outcome at individual DNA breakpoints.

Michael T Certo1, Byoung Y Ryu, James E Annis

  • 1Program in Molecular and Cellular Biology, University of Washington, Seattle, Washington, USA.

Nature Methods
|July 12, 2011
PubMed
Summary

This study introduces a novel genome engineering reporter system called "traffic light" for analyzing DNA repair pathways and optimizing gene editing outcomes in human cells. The system enables efficient isolation of cells with desired genetic modifications.

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Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy
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High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes
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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Site-specific genome engineering is crucial for biotechnological advancements.
  • Rare-cutting endonucleases are key tools for precise genome modification.
  • Understanding DNA break repair is essential for maximizing nuclease-based engineering.

Purpose of the Study:

  • To develop a reporter system for analyzing DNA repair pathway choice at nuclease-induced breaks.
  • To enable quantitative tracking of nuclease expression and donor template delivery.
  • To facilitate high-throughput screening for factors influencing genome engineering outcomes.

Main Methods:

  • Development of the 'traffic light' genome engineering reporter system.
  • Utilizing flow cytometry for rapid analysis of repair pathway choice.
  • Application of the system in human cell lines for evaluating engineering efficiency.

Main Results:

  • The 'traffic light' system allows for rapid, quantitative analysis of genome engineering events.
  • It facilitates the identification of strategies to improve the efficiency of desired genetic modifications.
  • The system was successfully applied to human cell lines, demonstrating its utility.

Conclusions:

  • The 'traffic light' system is a powerful tool for advancing site-specific genome engineering.
  • It provides insights into DNA repair mechanisms following nuclease-induced breaks.
  • This technology aids in the isolation of cells with precisely engineered genomes.