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Related Concept Videos

DNA Isolation01:24

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...

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Related Experiment Video

Updated: May 14, 2026

Automated Robotic Liquid Handling Assembly of Modular DNA Devices
11:22

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Published on: December 1, 2017

Ultra-low background DNA cloning system.

Kenta Goto1, Yukio Nagano

  • 1Analytical Research Center for Experimental Sciences, Saga University, Honjo, Saga, Japan. nagano@cc.saga-u.ac.jp

Plos One
|February 15, 2013
PubMed
Summary
This summary is machine-generated.

This study introduces an ultra-low background DNA cloning system using yeast recombination. The novel method significantly reduces by-products, achieving nearly 100% cloning efficiency for DNA fragments into E. coli vectors.

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Area of Science:

  • Molecular Biology
  • Synthetic Biology
  • Biotechnology

Background:

  • Yeast-based in vivo cloning utilizes homologous recombination for DNA fragment insertion into plasmid vectors.
  • While efficient, traditional yeast cloning methods generate undesirable by-products.
  • Escherichia coli (E. coli) vectors, particularly those with ampicillin resistance (Amp(r)), are widely used but can be challenging for yeast-based cloning.

Purpose of the Study:

  • To develop an "ultra-low background DNA cloning system" based on yeast in vivo cloning.
  • To significantly minimize or eliminate by-product generation during the cloning process.
  • To adapt the system for commonly used E. coli vectors, especially those lacking yeast replication origins.

Main Methods:

  • Construction of a specialized conversion cassette containing ampicillin resistance (Amp(r)) gene elements, yeast replication sequences (autonomous replication sequence and centromere), and a TRP1 yeast selectable marker.
  • Utilizing homologous recombination within yeast to convert E. coli vectors into yeast/E. coli shuttle vectors via the Amp(r) sequence.
  • Simultaneous transformation of the desired DNA fragment into yeast for direct cloning into the modified vector.

Main Results:

  • Rescued plasmid vectors from yeast transformants showed the complete absence of by-products containing the E. coli replication origin.
  • Subsequent transformation into E. coli eliminated by-products containing the yeast replication origin.
  • The developed system effectively uses yeast- and E. coli-specific origins of replication to purge unwanted DNA elements.
  • Successful cloning of DNA fragments into the vector was achieved with nearly 100% efficiency.

Conclusions:

  • The developed "ultra-low background DNA cloning system" effectively eliminates by-products by leveraging distinct replication origins in yeast and E. coli.
  • This method enhances the efficiency and purity of DNA cloning into E. coli vectors, overcoming limitations of traditional yeast-based approaches.
  • The system offers a robust and highly efficient solution for molecular cloning applications.