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Related Concept Videos

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...
The Replisome03:01

The Replisome

DNA replication is carried out by a large complex of proteins that act in a coordinated matter to achieve high-fidelity DNA replication. Together this complex is known as the DNA replication machinery or the replisome.
The synthesis of the leading and lagging strands is a highly coordinated process. To explain this, the “Trombone model” was proposed by Bruce Alberts in 1980. The DNA loop formation starts when a primer is synthesized on the parent lagging strand. The loop grows with 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...
Recombinant DNA01:09

Recombinant DNA

Overview
Next-generation Sequencing03:00

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
DNA Replication02:40

DNA Replication

DNA replication involves the separation of the two strands of the double helix, with each strand serving as a template from which the new complementary strand is copied.  After replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. This is known as semiconservative replication. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Replication in Prokaryotes
DNA replication uses a large number of...

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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

Constructing large DNA segments by iterative clone recombination.

Duane E Smailus1, Rene L Warren, Robert A Holt

  • 1Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Suite 100, 570 West 7th Avenue, Vancouver, BC, Canada, V5Z 4S6.

Systems and Synthetic Biology
|November 13, 2008
PubMed
Summary
This summary is machine-generated.

Scientists developed a novel vector system for assembling large DNA segments using in vivo recombination. This method efficiently constructs large DNA molecules, advancing synthetic biology applications.

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Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Genomics

Background:

  • Current DNA synthesis methods are limited in length (10s of kbp in vitro) or scale (100 kbp+ in vivo).
  • Efficient assembly of large DNA segments is crucial for synthetic biology applications like pathway engineering and whole-organism design.

Purpose of the Study:

  • To develop an efficient vector system for assembling large DNA molecules using iterative in vivo recombination.
  • To demonstrate the system's capability by reconstructing large genomic regions.

Main Methods:

  • Developed two custom fosmid vectors (pFOSAMP and pFOSKAN) enabling antibiotic switching.
  • Utilized iterative in vivo recombination of fosmid clones in a recombinogenic Escherichia coli host.
  • Reconstructed two non-contiguous genomic regions of Haemophilus influenzae as episomes.

Main Results:

  • Successfully assembled large DNA molecules using the developed vector system.
  • Reconstructed 190 kbp of the Haemophilus influenzae genome (10.4% of total genome) as episomes.
  • Demonstrated the efficiency of iterative in vivo recombination for large DNA assembly.

Conclusions:

  • The developed fosmid vector system enables efficient assembly of large DNA molecules via iterative in vivo recombination.
  • This method is a significant advancement for synthetic biology, facilitating the construction of complex genetic systems.
  • The technique successfully reconstructed substantial portions of a bacterial genome, showcasing its potential for large-scale DNA engineering.