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Related Concept Videos

FISH - Fluorescent In-situ Hybridization02:07

FISH - Fluorescent In-situ Hybridization

Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...
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...
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...
Gene Duplication and Divergence02:37

Gene Duplication and Divergence

The seminal work of Ohno in 1970 popularized the idea of gene duplication and divergence. DNA sequence comparison studies reveal that a large portion of the genes in bacteria, archaebacteria, and eukaryotes was  generated by gene duplication and divergence, indicating its critical role in evolution.
The duplicated copies of the gene are called Paralogs. Paralogs with similar sequences and functions form a gene family. Across several species, a large number of gene families are characterized.
Labeling DNA Probes03:31

Labeling DNA Probes

DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
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: Jun 15, 2026

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems
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Detecting variable (V), diversity (D) and joining (J) gene segment recombination using a two-colour fluorescence

Gina B Scott1, Erika A de Wynter, Graham P Cook

  • 1Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK. g.p.cook@leeds.ac.uk.

Mobile DNA
|March 16, 2010
PubMed
Summary

Researchers developed a novel two-color fluorescence system to easily detect V(D)J recombination events. This method simplifies the analysis of recombination activating gene (RAG)-mediated rearrangements without cytotoxic selection.

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Last Updated: Jun 15, 2026

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems
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Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae
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Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

Published on: September 11, 2022

Area of Science:

  • Immunology
  • Molecular Biology
  • Genetics

Background:

  • Adaptive immunity relies on immunoglobulin and T cell receptor diversity generated by V(D)J recombination.
  • Recombination activating gene (RAG) proteins mediate the rearrangement of V, D, and J gene segments for antigen recognition.
  • Traditional V(D)J recombination analysis uses extrachromosomal substrates and bacterial transformation, which can be cumbersome.

Purpose of the Study:

  • To develop a simplified, fluorescence-based system for detecting V(D)J recombination events.
  • To enable analysis of both deletion and inversion joining events mediated by RAG proteins.
  • To facilitate V(D)J recombination studies using integrated genomic substrates.

Main Methods:

  • A two-color fluorescence reporter system was designed using two distinct fluorescent reporter genes.
  • The system distinguishes unrearranged substrates from those undergoing RAG-mediated deletion or inversion.
  • Detection of recombination products is achieved via fluorescence microscopy or flow cytometry.

Main Results:

  • The fluorescence system effectively detects RAG-mediated deletion and inversion events.
  • Recombination products exhibit characteristics of true V(D)J recombination.
  • Analysis can be performed without the need for cytotoxic selection of products.
  • The system is compatible with substrates integrated into the genome.

Conclusions:

  • The developed fluorescence system simplifies the analysis and exploitation of the V(D)J recombination machinery.
  • This approach offers a more accessible method for studying RAG-mediated rearrangements.
  • Similar fluorescence-based strategies may be applicable to gene replacement during lymphocyte development.