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Related Concept Videos

Homologous Recombination02:31

Homologous Recombination

61.7K
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...
61.7K

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Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning
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High-Throughput DNA Assembly Using Yeast Homologous Recombination.

Kristy Ip1, Ron Yadin1, Kevin W George2

  • 1Amyris Inc., Emeryville, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|August 19, 2020
PubMed
Summary

Yeast homologous recombination offers a robust, cost-effective DNA assembly method. This protocol streamlines generating DNA parts and assembling them into vectors for high-throughput applications.

Keywords:
AutomationDNA assemblyHigh-throughputSynthetic biologyYeast homologous recombination

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Biotechnology

Background:

  • Yeast homologous recombination is a well-established technique for DNA assembly.
  • Existing methods can be time-consuming and costly for large-scale applications.

Purpose of the Study:

  • To describe a high-throughput protocol for DNA assembly using yeast homologous recombination.
  • To adapt the method for laboratory automation and large-scale DNA part generation.

Main Methods:

  • Generating DNA parts via Polymerase Chain Reaction (PCR).
  • Assembling multiple DNA parts into vectors using yeast transformation.
  • Shuttling assembled plasmid constructs into E. coli for propagation and storage.

Main Results:

  • Demonstrated efficient DNA assembly with short homology regions (as little as 24 base pairs).
  • Successfully assembled up to 12 unique DNA parts into various vectors.
  • Developed a protocol amenable to high-throughput workflows and laboratory automation.

Conclusions:

  • Yeast homologous recombination provides a reliable, efficient, and low-cost solution for DNA assembly.
  • The described high-throughput protocol can be adapted for both large-scale and bench-scale DNA assembly needs.
  • This method facilitates the generation and assembly of complex DNA constructs for diverse applications.