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Related Experiment Videos

Amplification of large artificial chromosomes.

D R Smith1, A P Smyth, D T Moir

  • 1Department of Human Genetics, Collaborative Research, Inc., Bedford, MA 01730.

Proceedings of the National Academy of Sciences of the United States of America
|November 1, 1990
PubMed
Summary

Yeast artificial chromosome (YAC) cloning is enhanced by a new system allowing copy number amplification. This method facilitates detailed genomic analysis by increasing YACs 10- to 20-fold without causing rearrangements.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Yeast artificial chromosome (YAC) cloning enables propagation of large DNA segments for genomic mapping.
  • Current limitations include low YAC copy number (one per haploid genome), necessitating extensive blotting-hybridization techniques for analysis.

Purpose of the Study:

  • To develop a YAC cloning vector and host strain system for controllable copy number amplification.
  • To improve the efficiency and scope of YAC-based genomic analyses.

Main Methods:

  • A novel YAC cloning vector featuring a conditional centromere regulated by carbon source was designed.
  • A heterologous thymidine kinase gene was incorporated for selection of amplified YACs.
  • Copy number amplification was induced and maintained under selective pressure.

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Main Results:

  • YACs ranging from 100 to 600 kilobases were amplified 10- to 20-fold.
  • Amplification occurred without inducing significant DNA rearrangements in tested clones.
  • Reactivation of the centromere allowed stable maintenance of elevated YAC copy numbers for at least 20 generations.

Conclusions:

  • The developed system effectively enables controllable copy number amplification of YACs.
  • This advancement significantly enhances the utility of YACs for detailed genomic studies and mapping.
  • The method offers a robust solution for overcoming low copy number limitations in YAC technology.