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Conservative Site-specific Recombination and Phase Variation02:53

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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.
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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...
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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...
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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. 
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Transposons make up a significant part of genomes of various organisms. Therefore, it is believed that transposition played a major evolutionary role in speciation by changing genome sizes and modifying gene expression patterns. For example, in bacteria, transposition can lead to conferring antibiotic resistance. Movement of transposable elements within the genetic pool of pathogenic bacteria can aid in transfer of antibiotic-resistant genetic elements. In eukaryotes, transposons can carry out...
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Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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Recombinase-free cloning (RFC) protocol for gene swapping.

Hai-Vy Vo-Nguyen1,2,3, Thanh-Tan Nguyen1,3, Quoc-Gia Mai1,2,3

  • 1Department Molecular and Environmental Biotechnology, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Vietnam.

Molecular Biology Research Communications
|April 25, 2022
PubMed
Summary

Recombinase-free cloning (RFC) simplifies DNA cloning by eliminating ligation and purification steps, using E. coli

Keywords:
CloningE.coli DH5αMolecular biologyRecombinant DNA

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetic Engineering

Background:

  • Recombinant DNA technology is crucial for molecular studies.
  • DNA ligases are essential for cloning but have limitations.
  • Existing cloning methods can be time-consuming and material-intensive.

Purpose of the Study:

  • To develop an improved DNA cloning method.
  • To simplify restriction enzyme-based cloning (REC).
  • To eliminate the need for DNA ligase and purification steps.

Main Methods:

  • Developed Recombinase-Free Cloning (RFC).
  • Utilized endogenous E. coli recombinase for in-cell vector creation.
  • Integrated PCR and digestion into a single temperature profile.
  • Eliminated all DNA purification steps.

Main Results:

  • Successfully generated five different DNA clones using RFC.
  • Achieved 100% success rate in trials with fragment sizes of 0.5-1.0 kbp.
  • Completed the RFC method within approximately 9 hours.
  • Demonstrated a material- and time-saving cloning process.

Conclusions:

  • RFC offers a more convenient and faster DNA cloning method.
  • The technique is suitable for laboratories with limited resources.
  • RFC significantly reduces the consumption of DNA components and materials.