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Related Concept Videos

Replication in Prokaryotes01:32

Replication in Prokaryotes

DNA replication has three main steps: initiation, elongation, and termination. Replication in prokaryotes begins when initiator proteins bind to the single origin of replication (ori) on the cell's circular chromosome. Replication then proceeds around the entire circle of the chromosome in each direction from the two replication forks, resulting in two DNA molecules.
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Homologous Recombination02:31

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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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Recombineering Homologous Recombination Constructs in Drosophila
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Published on: July 13, 2013

Recombineering linear DNA that replicate stably in E. coli.

Yaw-Shin Ooi1, Peter E Warburton, Nikolai V Ravin

  • 1Department of Genetics and Genomic Sciences, Box 1498, Mount Sinai School of Medicine, 1425 Madison Avenue, EB 14-02, New York, NY 10029, USA.

Plasmid
|November 9, 2007
PubMed
Summary

Researchers developed a new recombineering method to create linear DNA by adding telomeres from bacteriophage N15 to circular DNA. This technique produces stable, linear bacterial artificial chromosomes (BACs) resistant to degradation, useful for functional studies and cloning large genomes.

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Published on: January 8, 2015

Area of Science:

  • Molecular Biology
  • Genomics
  • Synthetic Biology

Background:

  • Recombineering in Escherichia coli offers advanced recombinant DNA construction.
  • Bacteriophage N15 exists as a linear prophage in E. coli, possessing unique telomeric structures.

Purpose of the Study:

  • To introduce a novel recombineering application for linearizing DNA using bacteriophage N15 telomeres.
  • To demonstrate the stable replication and resistance to degradation of telomere-capped linear DNA constructs.

Main Methods:

  • Recombination of the N15 telomerase occupancy site into circular DNA.
  • Resolution of telomeres using the phage N15 protelomerase enzyme.
  • Assembly and validation of linear bacterial artificial chromosomes (BACs) in E. coli DH10B.

Main Results:

  • Stable replication of linear BACs in E. coli DH10B confirmed by restriction mapping and pulsed field gel electrophoresis.
  • Linear BAC DNA demonstrated resistance to RecBCD nuclease degradation in vitro and in vivo.
  • Functional studies showed accurately spliced transcripts from transfected linear BACs in HT1080 cells.

Conclusions:

  • The novel recombineering technique enables the creation of stable, telomere-capped linear DNA.
  • This method is valuable for constructing large linear DNA for artificial chromosomes and cloning linear viral genomes.
  • Linear DNA constructs are suitable for subsequent functional studies and gene expression analysis.