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Related Experiment Videos

A versatile and general splitting technology for generating targeted YAC subclones.

Yeonhee Kim1, Minetaka Sugiyama, Kazuo Yamagishi

  • 1Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan.

Applied Microbiology and Biotechnology
|May 3, 2005
PubMed
Summary

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Improved yeast artificial chromosome (YAC) splitting technology enables subcloning of eukaryotic chromosome regions. This enhanced method successfully converted a targeted 60-kb region into a replicating YAC, overcoming limitations of the original technique.

Area of Science:

  • Molecular Biology
  • Genetics
  • Yeast Artificial Chromosomes (YACs)

Background:

  • Yeast artificial chromosomes (YACs) are crucial for cloning large DNA fragments.
  • Conventional YAC splitting technology allows subcloning of specific chromosomal regions.
  • Limitations exist in subcloning recalcitrant regions using the original YAC splitting method.

Purpose of the Study:

  • To improve YAC splitting technology for enhanced subcloning efficiency.
  • To develop a method for converting targeted eukaryotic chromosome regions within YACs into replicating YACs.
  • To demonstrate the efficacy of the improved YAC splitting technique on a difficult-to-clone region.

Main Methods:

  • Incorporation of PCR-mediated chromosome splitting and autonomously replicating sequences (ARS) into YAC technology.

Related Experiment Videos

  • Design and synthesis of specific splitting fragments using template plasmids (pSK-KCA, pSKCLY).
  • Introduction of splitting fragments into Saccharomyces cerevisiae harboring a split YAC containing the target region.

    • Main Results:

    • A 60-kb region from a 590-kb YAC was successfully split and converted into a replicating YAC.
    • Four out of 12 Leu(+) transformants showed the expected karyotype with newly generated 40-kb and 60-kb chromosomes.
    • The improved method demonstrated its capability to isolate and replicate specific chromosomal segments.

    Conclusions:

    • The enhanced YAC splitting technology effectively subclones targeted eukaryotic chromosome regions.
    • This improved method overcomes previous limitations in YAC manipulation.
    • The technique facilitates the creation of novel replicating YACs from specific chromosomal segments.