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A versatile genome-scale PCR-based pipeline for high-definition DNA FISH

Abstract

We developed a cost-effective genome-scale PCR-based method for high-definition DNA FISH (HD-FISH). We visualized gene loci with diffraction-limited resolution, chromosomes as spot clusters and single genes together with transcripts by combining HD-FISH with single-molecule RNA FISH. We provide a database of over 4.3 million primer pairs targeting the human and mouse genomes that is readily usable for rapid and flexible generation of probes.

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Figure 1: HD-FISH probe design and synthesis.
Figure 2: Specificity and sensitivity of HD-FISH.
Figure 3: Versatility of HD-FISH.

References

  1. Springer Protocols. Fluorescence In Situ Hybridization (FISH)–Application Guide (ed. Liehr, T.) (Springer, 2008).

  2. Springer Protocols. Fluorescence In Situ Hybridization (FISH): Protocols and Applications (Methods in Molecular Biology). (eds Bridger, J.M. & Volpi, E.V.) (Humana Press, 2010).

  3. Boyle, S., Rodesch, M.J., Halvensleben, H.A., Jeddeloh, J.A. & Bickmore, W.A. Chromosome Res. 19, 901–909 (2011).

    CAS  Article  Google Scholar 

  4. Yamada, N.A. et al. Cytogenet. Genome Res. 132, 248–254 (2011).

    CAS  Article  Google Scholar 

  5. Rogan, P.K., Cazcarro, P.M. & Knoll, J.H. Genome Res. 11, 1086–1094 (2001).

    CAS  Article  Google Scholar 

  6. Navin, N. et al. Bioinformatics 22, 2437–2438 (2006).

    CAS  Article  Google Scholar 

  7. Wiegant, J.C. et al. Cytogenet. Cell Genet. 87, 47–52 (1999).

    CAS  Article  Google Scholar 

  8. Solovei, I. & Cremer, M. Methods Mol. Biol. 659, 117–126 (2010).

    CAS  Article  Google Scholar 

  9. Cremer, T. & Cremer, M. Cold Spring Harb. Perspect. Biol. 2, a003889 (2010).

    Article  Google Scholar 

  10. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Nat. Methods 5, 877–879 (2008).

    CAS  Article  Google Scholar 

  11. Tsafrir, D. et al. Cancer Res. 66, 2129–2137 (2006).

    CAS  Article  Google Scholar 

  12. Beliveau, B.J. et al. Proc. Natl. Acad. Sci. USA published online, doi:10.1073/pnas.1213818110 (2012).

  13. Kent, W.J. Genome Res. 12, 656–664 (2002).

    CAS  Article  Google Scholar 

  14. Untergasser, A. et al. Nucleic Acids Res. 40, e115 (2012).

    CAS  Article  Google Scholar 

  15. Wheeler, D.L. et al. Nucleic Acids Res. 31, 28–33 (2003).

    CAS  Article  Google Scholar 

  16. Itzkovitz, S. et al. Nat. Cell Biol. 14, 106–114 (2012).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank P. Junker and S. Semrau for helpful discussions. We are grateful to R.A. Weinberg (Massachusetts Institute of Technology) for providing hTERT-HME1 cells. This work was supported by the US National Institutes of Health (NIH)/National Cancer Institute Physical Sciences Oncology Center at Massachusetts Institute of Technology (U54CA143874), an NIH Pioneer award (1DP1OD003936) and a Nederlandse Organisatie voor Wetenschappelijk Onderzoek Vici award to A.v.O. M.B. and S.I. are sponsored by the Human Frontiers Science Program.

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Authors and Affiliations

Authors

Contributions

N.C. and A.v.O. conceived the methods. M.B. and N.C. performed experiments, analyzed the data and wrote the manuscript. L.T. generated the genome-wide primer databases, designed the probes and wrote the manuscript. S.K. and S.I. developed software for image processing, provided suggestions on data analysis and corrected the manuscript. A.v.O. guided experiments and data analysis, and wrote the manuscript.

Corresponding author

Correspondence to Alexander van Oudenaarden.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Note (PDF 9821 kb)

3D rendering of Chr17 in HME cells, visualized with ten HD-FISH probes evenly spaced every 8 Mb and labeled with two alternating fluorophores (green: AlexaFluor594; magenta: AlexaFluor647).

The nucleus displayed is the same as in the Z-projection shown in Figure 3a (mid panel). (MOV 5461 kb)

41592_2013_BFnmeth2306_MOESM170_ESM.mov

3D animation of Chr17 in HME cells, visualized with sixteen HD-FISH probes spaced evenly every 5 Mb and labeled with two alternating fluorophores (green: AlexaFluor594; magenta: AlexaFluor647) together with a Chr17 paint probe (blue). (MOV 4960 kb)

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Bienko, M., Crosetto, N., Teytelman, L. et al. A versatile genome-scale PCR-based pipeline for high-definition DNA FISH. Nat Methods 10, 122–124 (2013). https://doi.org/10.1038/nmeth.2306

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  • DOI: https://doi.org/10.1038/nmeth.2306

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