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Isolation of circular yeast artificial chromosomes for synthetic biology and functional genomics studies

Nature Protocols volume 6, pages 89–96 (2011)Cite this article

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Abstract

Circular yeast artificial chromosomes (YACs) provide significant advantages for cloning and manipulating large segments of genomic DNA in Saccharomyces cerevisiae. However, it has been difficult to exploit these advantages, because circular YACs are difficult to isolate and purify. Here we describe a method for purification of large circular YACs that is more reliable compared with previously described protocols. This method has been used to purify YACs up to 600 kb in size. The purified YAC DNA is suitable for restriction enzyme digestion, DNA sequencing and functional studies. For example, YACs carrying full-size genes can be purified from yeast and used for transfection into mammalian cells or for the construction of a synthetic genome that can be used to produce a synthetic cell. This method for isolating high-quality YAC DNA in microgram quantities should be valuable for functional and synthetic genomic studies. The entire protocol takes ∼3 d to complete.

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Figure 1: Purification of circular YAC from 400 ml of yeast culture.
Figure 2: Topological trapping of circular YACs in agarose plugs.

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Acknowledgements

The research reported in this article was supported by Synthetic Genomics (SGI) (V.N.N., R.-Y.C. and D.G.G.) and by the intramural research program of the National Institutes of Health National Cancer Institute, Center for Cancer Research (V.L. and N.K.).

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Authors and Affiliations

  1. The J. Craig Venter Institute, Rockville, Maryland, USA

    Vladimir N Noskov, Ray-Yuan Chuang & Daniel G Gibson

  2. Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA

    Sun-Hee Leem, Vladimir Larionov & Natalay Kouprina

  3. Department of Biological Science, Dong-A University, Busan, Korea

    Sun-Hee Leem

Authors
  1. Vladimir N Noskov
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  2. Ray-Yuan Chuang
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  3. Daniel G Gibson
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  4. Sun-Hee Leem
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  5. Vladimir Larionov
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  6. Natalay Kouprina
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Contributions

V.N.N. and R.-Y.C. optimized and tested the protocols, generated the figures and cowrote the Procedure and Materials sections. D.G.G. developed topological trapping of circular DNA and worked on Troubleshooting. S.-H.L. suggested using the Qiagen construct kit for YAC purification. V.L. and N.K. suggested the use of alkaline lysis for large circular DNA purification from yeast and worked on the Abstract, Introduction, Experimental design and References sections.

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Correspondence to Ray-Yuan Chuang or Natalay Kouprina.

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The authors declare no competing financial interests.

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Noskov, V., Chuang, RY., Gibson, D. et al. Isolation of circular yeast artificial chromosomes for synthetic biology and functional genomics studies. Nat Protoc 6, 89–96 (2011). https://doi.org/10.1038/nprot.2010.174

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