Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Microfluidic-assisted analysis of replicating DNA molecules

Nature Protocols volume 4, pages 849–861 (2009)Cite this article

Abstract

Single molecule-based protocols have been gaining popularity as a way to visualize DNA replication at the global genomic- and locus-specific levels. These protocols take advantage of the ability of many organisms to incorporate nucleoside analogs during DNA replication, together with a method to display stretched DNA on glass for immunostaining and microscopy. We describe here a microfluidic platform that can be used to stretch and to capture labeled DNA molecules for replication analyses. This platform consists of parallel arrays of three-sided, 3- or 4-μm high, variable-width capillary channels fabricated from polydimethylsiloxane by conventional soft lithography, and of silane-modified glass coverslips to reversibly seal the open side of the channels. Capillary tension in these microchannels facilitates DNA loading, stretching and glass coverslip deposition from microliter-scale DNA samples. The simplicity and extensibility of this platform should facilitate DNA replication analyses using small samples from a variety of biological and clinical sources.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Overview of a setup for microchannel-assisted replication track analysis (maRTA).
Figure 2: Fabrication of a 'negative master' for casting PDMS microchannels using standard photolithography procedures.
Figure 3: Fabrication of PDMS capillary microchannels by soft lithography.
Figure 4: Derivatizing glass coverslips for DNA capture, immunostaining and microscopy.
Figure 5: Examples of stretched DNA.
Figure 6: Replication track types and measures that can be derived from track images.
Figure 7: Stretching parameters.

Similar content being viewed by others

References

  1. Alberts, B. DNA replication and recombination. Nature 421, 431–435 (2003).

    Article  PubMed  Google Scholar 

  2. Kelly, T. & Stillman, B. Duplication of DNA in eukaryotic cells. In DNA Replication and Human Disease (ed. DePamphilis, M.L.) 1–29 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2006).

    Google Scholar 

  3. Loeb, L.A. & Monnat, R.J. DNA polymerases and human disease. Nat. Rev. Genet. 9, 594–604 (2008).

    CAS  PubMed  Google Scholar 

  4. McCulloch, S.D. & Kunkel, T.A. The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases. Cell Res. 18, 148–161 (2008).

    Article  CAS  PubMed  Google Scholar 

  5. Yang, W. & Woodgate, R. What a difference a decade makes: insights into translesion DNA synthesis. Proc. Natl. Acad. Sci. USA 104, 15591–15598 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gilson, E. & Geli, V. How telomeres are replicated. Nat. Rev. Mol. Cell Biol. 8, 825–838 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Bianchi, A. & Shore, D. How telomerase reaches its end: mechanism of telomerase regulation by the telomeric complex. Mol. Cell 31, 153–165 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Kornberg, A. & Baker, T.A. DNA Replication (W.H. Freeman, New York, 1991).

    Google Scholar 

  9. Pommier, Y. & Diasio, R.B. Pharmacologic agents that target DNA replication. In DNA Replication and Human Disease (ed. DePamphilis, M.L.) 519–546 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 2006).

    Google Scholar 

  10. Berdis, A.J. DNA polymerases as therapeutic targets. Biochemistry 47, 8253–8260 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Vengrova, S. & Dalgaard, J.Z. Eds. DNA Replication Methods and Protocols (Humana Press, New York, 2009).

    Book  Google Scholar 

  12. Manders, E.M., Stap, J., Brakenhoff, G.J., van Driel, R. & Aten, J.A. Dynamics of three-dimensional replication patterns during the S-phase, analysed by double labelling of DNA and confocal microscopy. J. Cell Sci. 103, 857–862 (1992).

    CAS  PubMed  Google Scholar 

  13. Jackson, D.A. & Pombo, A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J. Cell Biol. 140, 1285–95 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Huberman, J.A. & Riggs, A.D. Autoradiography of chromosomal DNA fibers from Chinese hamster cells. Proc. Natl. Acad. Sci. USA 55, 599–606 (1966).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Cairns, J. The chromosome of Escherichia coli . In Cold Spring Harbor Symposia on Quantitative Biology Vol. 28 43–46 (Cold Spring Harbor Press, Cold Spring Harbor, New York, 1963).

    Google Scholar 

  16. Bensimon, A. et al. Alignment and sensitive detection of DNA by a moving interface. Science 265, 2096–2098 (1994).

    Article  CAS  PubMed  Google Scholar 

  17. Yokota, H. et al. A new method for straightening DNA molecules for optical restriction mapping. Nucleic Acids Res. 25, 1064–1070 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Herrick, J. & Bensimon, A. Single molecule analysis of DNA replication. Biochimie 81, 859–871 (1999).

    Article  CAS  PubMed  Google Scholar 

  19. Norio, P. & Schildkraut, C.L. Visualization of DNA replication on individual Epstein–Barr virus episomes. Science 294, 2361–2364 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Sidorova, J.M., Li, N., Folch, A. & Monnat, R.J. Jr. The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest. Cell Cycle 7, 796–807 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Dimalanta, E.T. et al. A microfluidic system for large DNA molecule arrays. Anal. Chem. 76, 5293–5301 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Zhou, S., Herschleb, J. & Schwartz, D.C. A single molecule system for whole genome analysis. In New High Throughput Technologies for DNA Sequencing and Genomics (ed. Mitchelson, K.R.) 265–302 (Elsevier, Amsterdam, 2007).

    Chapter  Google Scholar 

  23. Zhou, S. et al. Validation of rice genome sequence by optical mapping. BMC Genomics 8, 278 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Valouev, A., Schwartz, D., Zhou, S. & Waterman, M.S. An algorithm for assembly of ordered restriction maps from single DNA molecules. Proc. Natl. Acad. Sci. USA 103, 15770–15775 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kidd, J.M. et al. Mapping and sequencing of structural variation from eight human genomes. Nature 453, 56–64 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. McDonald, J.C. et al. Fabrication of microfluidic systems in poly(dimethylsiloxane). Electrophoresis 21, 27–40 (2000).

    Article  CAS  PubMed  Google Scholar 

  27. Beebe, D.J., Mensing, G.A. & Walker, G.M. Physics and applications of microfluidics in biology. Annu. Rev. Biomed. Eng. 4, 261–286 (2002).

    Article  CAS  PubMed  Google Scholar 

  28. Tegenfeldt, J. et al. Micro- and nanofluidics for DNA analysis. Anal. Bioanal. Chem. 378, 1678–1692 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Atencia, J. & Beebe, D.J. Controlled microfluidic interfaces. Nature 437, 648–655 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Whitesides, G.M. The origins and the future of microfluidics. Nature 442, 368–373 (2006).

    Article  CAS  PubMed  Google Scholar 

  31. Herschleb, J., Ananiev, G. & Schwartz, D.C. Pulsed-field gel electrophoresis. Nat. Protoc. 2, 677–684 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Lundin, C. et al. Methyl methanesulfonate (MMS) produces heat-labile DNA damage but no detectable in vivo DNA double-strand breaks. Nucleic Acids Res. 33, 3799–3811 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the NIH Seattle Cancer and Aging Program Grant P20 CA103728Subaward 000618167 to J.S., an NHGRI R01 (HG000225) to D.C.S., an R21/R33 EB0003307 award to A.F. and NCI PO1 CA88752 to R.J.M. Jr. We also thank Dhalia Dhingra and Paolo Norio for advice during early phases of this work, Amelia Gallaher for technical assistance and Alden Hackmann for graphics support.

Author information

Author notes

  1. Nianzhen Li

    Present address: Present address: Fluxion Biosciences, South San Francisco, California, USA.,

Authors and Affiliations

  1. Department of Pathology, University of Washington, Seattle, Washington, USA

    Julia M Sidorova & Raymond J Monnat Jr

  2. Department of Bioengineering, University of Washington, Seattle, Washington, USA

    Nianzhen Li & Albert Folch

  3. Departments of Genetics and Chemistry and UW-Biotechnology Center, Laboratory for Molecular and Computational Genomics, University of Wisconsin-Madison, Madison, Wisconsin, USA

    David C Schwartz

  4. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    David C Schwartz & Raymond J Monnat Jr

Authors
  1. Julia M Sidorova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nianzhen Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David C Schwartz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Albert Folch
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Raymond J Monnat Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Julia M Sidorova.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sidorova, J., Li, N., Schwartz, D. et al. Microfluidic-assisted analysis of replicating DNA molecules. Nat Protoc 4, 849–861 (2009). https://doi.org/10.1038/nprot.2009.54

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.54

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing