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Related Concept Videos

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...
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...
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...
Mismatch Repair01:36

Mismatch Repair

Overview
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...

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Related Experiment Video

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Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Published on: January 8, 2015

Using recombineering to generate point mutations: the oligonucleotide-based "hit and fix" method.

Suhwan Chang1, Stacey Stauffer, Shyam K Sharan

  • 1Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA.

Methods in Molecular Biology (Clifton, N.J.)
|February 14, 2012
PubMed
Summary

This study introduces a two-step "Hit and Fix" recombineering method for efficient genome manipulation. This technique facilitates precise gene editing and functional studies in bacteria using bacteriophage lambda systems.

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Recombineering Homologous Recombination Constructs in Drosophila
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Recombineering Homologous Recombination Constructs in Drosophila

Published on: July 13, 2013

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Last Updated: May 25, 2026

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
09:02

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Published on: January 8, 2015

Recombineering Homologous Recombination Constructs in Drosophila
14:23

Recombineering Homologous Recombination Constructs in Drosophila

Published on: July 13, 2013

Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • Genome manipulation is essential for understanding gene function.
  • Recombineering offers an efficient method for large-scale genetic modifications.
  • Bacteriophage lambda-based systems provide robust recombination machinery.

Purpose of the Study:

  • To develop a novel, efficient two-step method for precise genome editing.
  • To enhance the generation of subtle genetic alterations in bacterial DNA.
  • To facilitate screening of recombinant clones for functional studies.

Main Methods:

  • Utilized a two-step "Hit and Fix" recombineering strategy.
  • Employed bacteriophage lambda-derived genes (exo, bet, gam) for recombination.
  • Inserted a temporary heterologous sequence (Hit) followed by replacement with desired mutation (Fix).
  • Screened recombinant clones using Polymerase Chain Reaction (PCR) and colony hybridization.

Main Results:

  • Demonstrated high efficiency in generating subtle genetic alterations.
  • Successfully applied the method to both chromosomal and episomal DNA.
  • Enabled straightforward screening of desired genetic modifications.

Conclusions:

  • The "Hit and Fix" method provides a powerful tool for precise genome engineering.
  • This approach significantly improves the feasibility and efficiency of functional genomic studies.
  • Recombineering, particularly with this novel strategy, is highly effective for targeted DNA modifications.