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High resolution DNA Fiber–fish on yeast artificial chromosomes: direct visualization of DNA replication

Nature Genetics volume 10, pages 477–479 (1995)Cite this article

An Erratum to this article was published on 01 September 1995

Abstract

Fluorescent in situ hybridization (FISH) is a powerful, direct and sensitive technique with a wide resolution range that enables the simultaneous study of multiple targets, labelled in different colours. Spreading techniques, denoted here as ‘Fiber-FISH’, increase FISH-resolution to the DMA fiber, using decondensed nuclear DMA as hybridization target1–5. FISH could be a powerful analytical tool for thorough physical examination of yeast artificial chromosomes (YACs) which are often chimaeric or contain internal deletions. However, with one exception restricted to meiotic yeast chromosomes6, FISH has not been used successfully on yeast/YAC DMA. We have developed a fast and simple method that can be applied routinely for compositional and structural analysis of cosmid and YAC DMA in yeast. It enables precise localization and ordering of clones, resolves overlaps and distances and gives a detailed picture of the integrity and colinearity of both probe and target. The combination of high resolution, signal abundance and short yeast cell cycle allows direct visualization of replicating DMA fibers. In a 400 kb region of the human dystrophin gene, we identified two replication origins, demonstrating that human DMA cloned in yeast is capable of initiating its own replication.

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References

  1. Heng, H.H.Q., Squire, J. & Tsui, L.C. High resolution mapping af mammalian genes by in situ hybridization to free chromatin. Proc. natn. Acad. Sci. U.S.A. 89, 9509–9513 (1992).

    Article  CAS  Google Scholar 

  2. Wiegant, J. et al. High-resolution in situ hybridization using DNA halo preparations. Hum. molec. Genet. 1, 587–591 (1992).

    Article  CAS  Google Scholar 

  3. Parra, I. & Windle, B. High resolution visual mapping of stretched DNA by fluorescent hybridization. Nature Genet. 5, 17–21 (1993).

    Article  CAS  Google Scholar 

  4. Haaf, T. & Ward, D.C. High resolution ordering of YAC contigs using extended chromatin and chromosomes. Hum. molec. Genet. 3, 629–633 (1994).

    Article  CAS  Google Scholar 

  5. Heiskanen, M., Karhu, R., Hellsten, E., Peltonen, L., Kallioniemi, O.P. & Palotie, A. High resolution mapping using fluorescence in situ hybridization to extended DNA fibers prepared from agarose-embedded cells. Bio Techniques 17, 928–933 (1994).

    CAS  Google Scholar 

  6. Scherthan, H., Schweizer, D. & Loidl, J. Delineation of individual chromosomes of Saccharomyces cerevisiae by two-colour in situ hybridization. Trends Genet. 9, 41 (1993).

    Article  CAS  Google Scholar 

  7. Florijn, R.J. et al. High-resolution FISH for genomic DNA mapping and colour bar-coding of large genes. Hum. molec. Genet. 4, 831–836 (1995).

    Article  CAS  Google Scholar 

  8. Blonden, L.A.J. et al. 242 breakpoints in the 200-kb deletion-prone P20 region of the DMD-gene are widely spread. Genomics 10, 631–639 (1991).

    Article  CAS  Google Scholar 

  9. Roth, G.E., Blanton, H.M. & Zakian, V.A. Isolation and characterization of sequences from mouse chromosomal DNA with ARS function in yeast. Molec. cell. Biol. 3, 1898–1908 (1983).

    Article  CAS  Google Scholar 

  10. Newlon, C.S. Yeast chromosome replication and segregation. Microbiol. Rev. 52, 568–601 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Monteil, J.F. et al. Characterization of human chromosomal DNA sequences which replicate autonomously in Saccharomyces cerevisiae. Nucl. Acids Res. 12, 1049–1068 (1984).

    Article  Google Scholar 

  12. Edenberg, H.J. & Huberman, J.A. Eukaryotic DNA replication. Annu. Rev. Genet. 9, 245–284 (1975).

    Article  CAS  Google Scholar 

  13. Selig, S., Okumura, K., Ward, D.C. & Cedar, H. Delineation of DNA replication time zones by fluorescence in situ hybridization. EMBO J. 11, 1217–1225 (1992).

    Article  CAS  Google Scholar 

  14. Kitsberg, D., Selig, S., Keshet, I. & Cedar, H. Replication structure of the beta-globin gene domain. Nature 366, 588–590 (1993).

    Article  CAS  Google Scholar 

  15. Koenig, M. et al. Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50, 509–517 (1987).

    Article  CAS  Google Scholar 

  16. Den Dunnen, J.T. et al. Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. Hum. molec. Genet 1, 19–28 (1992).

    Article  CAS  Google Scholar 

  17. Kievits, T. et al. Rapid sub-chromosomal localization of cosmids by non-radioactive in situ hybridization. Cytogenet. Cell Genet. 53, 134–136 (1990).

    Article  CAS  Google Scholar 

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Authors
  1. Carla Rosenberg
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  2. Ralph J. Florijn
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  3. Frans M. Van De Rijke
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  4. Lau A.J. Blonden
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  5. Ton K. Raap
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  6. Gert-Jan B. Van Ommen
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  7. Johan T. Den Dunnen
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Rosenberg, C., Florijn, R., Van De Rijke, F. et al. High resolution DNA Fiber–fish on yeast artificial chromosomes: direct visualization of DNA replication. Nat Genet 10, 477–479 (1995). https://doi.org/10.1038/ng0895-477

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