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Fluorescence Microscopy of Nanochannel-Confined DNA

  • Fredrik Persson
  • Fredrik Westerlund
  • Jonas O. TegenfeldtEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 783)

Abstract

Stretching of DNA in nanoscale confinement allows for direct visualization of the genetic contents of the DNA on the single DNA molecule level. DNA stretched in nanoscale confinement also allows for studies of DNA–protein interactions and DNA polymer physics in confined environments. This chapter describes the basic steps to fabricate the nanostructures, to perform the experiments, and to analyze the data.

Key words

DNA Nanochannels Single molecule Fluorescence 

Notes

Acknowledgments

We thank Camilla Freitag for valuable discussions and comments on the manuscript.

This work was supported by the European Community’s Seventh Framework Program [FP7/2007–2013] under grant agreement number [HEALTH-F4–2008–201418] entitled READNA, the Swedish Research Council under grants number 2007-584 and 2007-4454 and the Knut and Alice Wallenberg Foundation.

References

  1. 1.
    Tegenfeldt, J. O., Prinz, C., Cao, H., Chou, S., Reisner, W. W., Riehn, R., Wang, Y. M., Cox, E. C., Sturm, J. C., Silberzan, P., and Austin, R. H. (2004) The dynamics of genomic-length DNA molecules in 100-nm channels, Proceedings of the National Academy of Sciences of the United States of America 101, 10979–10983.PubMedCrossRefGoogle Scholar
  2. 2.
    Wang, Y. M., Tegenfeldt, J. O., Reisner, W., Riehn, R., Guan, X. J., Guo, L., Golding, I., Cox, E. C., Sturm, J., and Austin, R. H. (2005) Single-molecule studies of repressor-DNA interactions show long-range interactions, Proceedings of the National Academy of Sciences of the United States of America 102, 9796–9801.PubMedCrossRefGoogle Scholar
  3. 3.
    Riehn, R., Lu, M. C., Wang, Y. M., Lim, S. F., Cox, E. C., and Austin, R. H. (2005) Restriction mapping in nanofluidic devices, Proceedings of the National Academy of Sciences of the United States of America 102, 10012–10016.PubMedCrossRefGoogle Scholar
  4. 4.
    Reisner, W., Morton, K. J., Riehn, R., Wang, Y. M., Yu, Z. N., Rosen, M., Sturm, J. C., Chou, S. Y., Frey, E., and Austin, R. H. (2005) Statics and dynamics of single DNA molecules confined in nanochannels, Physical Review Letters 94, 196101.PubMedCrossRefGoogle Scholar
  5. 5.
    Persson, F., Westerlund, F., Tegenfeldt, J. O., and Kristensen, A. (2009) Local Conformation of Confined DNA Studied Using Emission Polarization Anisotropy, Small 5, 190–193.PubMedCrossRefGoogle Scholar
  6. 6.
    Persson, F., Utko, P., Reisner, W., Larsen, N. B., and Kristensen, A. (2009) Confinement Spectroscopy: Probing Single DNA Molecules with Tapered Nanochannels, Nano Letters 9, 1382–1385.PubMedCrossRefGoogle Scholar
  7. 7.
    Stankiewicz, P., and Lupski, J. R. (2002) Genome architecture, rearrangements and genomic disorders, Trends in Genetics 18, 74–82.PubMedCrossRefGoogle Scholar
  8. 8.
    Vologodskii, A., and Cozzarelli, N. (1995) Modeling of long-range electrostatic interactions in DNA, Biopolymers 35, 289–296.PubMedCrossRefGoogle Scholar
  9. 9.
    Daoud, M., and de Gennes, P. G. (1977) Statistics of macromolecular solutions trapped in small pores, Le Journal de Physique 38, 85–93.CrossRefGoogle Scholar
  10. 10.
    Turban, L. (1984) Conformation of Confined Macromolecular Chains - Crossover Between Slit and Capillary, Journal De Physique 45, 347–353.CrossRefGoogle Scholar
  11. 11.
    Zhang, C., Zhang, F., van Kan, J. A., and van der Maarel, J. R. C. (2008) Effects of electrostatic screening on the conformation of single DNA molecules confined in a nanochannel, Journal of Chemical Physics 128.Google Scholar
  12. 12.
    Hsieh, C. C., and Doyle, P. S. (2008) Studying confined polymers using single-molecule DNA experiments, Korea-Australia Rheology Journal 20, 127–142.Google Scholar
  13. 13.
    Persson, F., and Tegenfeldt, J. O. (2010) DNA in nanochannels - directly visualizing genomic information, Chemical Society Reviews 39, 985–999.PubMedCrossRefGoogle Scholar
  14. 14.
    de Gennes, P. G. (1979) Scaling Concepts in Polymer Physics, Cornell University Press, Ithaca, NY.Google Scholar
  15. 15.
    Doi, M., and Edwards, S. F. (1986) The Theory of Polymer Dynamics, Vol. 73, Oxford University Press, Inc., New York.Google Scholar
  16. 16.
    Rubinstein, M., and Colby, R. H. (2003) Polymer Physics, Oxford University Press, New York.Google Scholar
  17. 17.
    Madou, M. J. (2002) Fundamentals of Microfabrication: the science of miniaturization, 2 ed., CRC Press LLC.Google Scholar
  18. 18.
    Mertz, J. (2010) Introduction to optical microscopy, Roberts and Company Publishers, Greenwood Village.Google Scholar
  19. 19.
    Persson, F. (2009) Nanofluidics for Single Molecule in DTU Nanotech - Department of Micro- and Nanotechnology, Technical University of Denmark, Kongens Lyngby.Google Scholar
  20. 20.
    Reisner, W., Beech, J. P., Larsen, N. B., Flyvbjerg, H., Kristensen, A., and Tegenfeldt, J. O. (2007) Nanoconfinement-enhanced conformational response of single DNA molecules to changes in ionic environment, Physical Review Letters 99, 058302.PubMedCrossRefGoogle Scholar
  21. 21.
    Han, J., Turner, S. W., and Craighead, H. G. (1999) Entropic trapping and escape of long DNA molecules at submicron size constriction, Physical Review Letters 83, 1688–1691.CrossRefGoogle Scholar
  22. 22.
    Quake, S. R., Babcock, H., and Chu, S. (1997) The dynamics of partially extended single molecules of DNA, Nature 388, 151–154.PubMedCrossRefGoogle Scholar
  23. 23.
    Glazer, A. N., and Rye, H. S. (1992) Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection, Nature 359, 859–861.PubMedCrossRefGoogle Scholar
  24. 24.
    Spielmann, H. P., Wemmer, D. E., and Jacobsen, J. P. (1995) Solution Structure of a DNA Complex with the Fluorescent Bis-Intercalator TOTO Determined by NMR-Spectroscopy, Biochemistry 34, 8542–8553.PubMedCrossRefGoogle Scholar
  25. 25.
    Lerman, L. S. (1961) Structural Considerations in Interaction of DNA and Acridines, J. Mol. Biol. 3, 18–30.PubMedCrossRefGoogle Scholar
  26. 26.
    Reinert, K. E. (1973) DNA stiffening and elongation caused by binding of ethidium bromide, Biochimica Et Biophysica Acta 319, 135–139.PubMedGoogle Scholar
  27. 27.
    Murade, C. U., Subramaniam, V., Otto, C., and Bennink, M. L. (2009) Interaction of Oxazole Yellow Dyes with DNA Studied with Hybrid Optical Tweezers and Fluorescence Microscopy, Biophysical Journal 97, 835–843.PubMedCrossRefGoogle Scholar
  28. 28.
    Thamdrup, L. H., Persson, F., Bruus, H., Kristensen, A., and Flyvbjerg, H. (2007) Experimental investigation of bubble formation during capillary filling of SiO2 nanoslits, Applied Physics Letters 91, 163505.CrossRefGoogle Scholar
  29. 29.
    Thompson, R. E., Larson, D. R., and Webb, W. W. (2002) Precise nanometer localization analysis for individual fluorescent probes, Biophysical Journal 82, 2775–2783.PubMedCrossRefGoogle Scholar
  30. 30.
    Huang, B., Bates, M., and Zhuang, X. (2009) Super-Resolution Fluorescence Microscopy, Annual Review of Biochemistry 78, 993–1016.PubMedCrossRefGoogle Scholar
  31. 31.
    Perkins, T. T., Smith, D. E., and Chu, S. (1997) Single polymer dynamics in an elongational flow, Science 276, 2016–2021.PubMedCrossRefGoogle Scholar
  32. 32.
    Morton, K. J., Loutherback, K., Inglis, D. W., Tsui, O. K., Sturm, J. C., Chou, S. Y., and Austin, R. H. (2008) Crossing microfluidic streamlines to lyse, label and wash cells, Lab on a Chip 8, 1448–1453.PubMedCrossRefGoogle Scholar
  33. 33.
    Persson, F., Thamdrup, L. H., Mikkelsen, M. B. L., Jaarlgard, S. E., Skafte-Pedersen, P., Bruus, H., and Kristensen, A. (2007) Double thermal oxidation scheme for the fabrication of SiO2 nanochannels, Nanotechnology 18, 245301.CrossRefGoogle Scholar
  34. 34.
    Riehn, R., and Austin, R. H. (2006) Wetting micro- and nanofluidic devices using supercritical water, Analytical Chemistry 78, 5933–5934.PubMedCrossRefGoogle Scholar
  35. 35.
    Elf, J., Li, G. W., and Xie, X. S. (2007) Probing transcription factor dynamics at the single-molecule level in a living cell, Science 316, 1191–1194.PubMedCrossRefGoogle Scholar
  36. 36.
    Persson, F. and Tegenfeldt, J. O. (2010) DNA in nanochannels - directly visualizing genomic information, Chem Soc Reviews, 39, 985–999.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Fredrik Persson
    • 1
  • Fredrik Westerlund
    • 1
  • Jonas O. Tegenfeldt
    • 1
    • 2
    Email author
  1. 1.Department of PhysicsUniversity of GothenburgGothenburgSweden
  2. 2.Department of PhysicsLund UniversityLundSweden

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