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.

  • Perspective
  • Published:

Laminar flow cells for single-molecule studies of DNA-protein interactions

Nature Methods volume 5, pages 517–525 (2008)Cite this article

Abstract

Microfluidic flow cells are used in single-molecule experiments, enabling measurements to be made with high spatial and temporal resolution. We discuss the fundamental processes affecting flow cell operation and describe the flow cells in use at present for studying the interaction of optically trapped or mechanically isolated, single DNA molecules with proteins. To assist the experimentalist in flow cell selection, we review the construction techniques and materials used to fabricate both single- and multiple-channel flow cells and the advantages of each design for different experiments.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Fluid flow in multistream laminar flow cells.
Figure 2: A simple single-stream flow cell.
Figure 3: A single-stream flow cell for force measurements.
Figure 4: Multistream laminar flow cell for single-molecule studies.
Figure 5: Multistream flow cells facilitate careful single-molecule experiments.
Figure 6: A laminar boundary-steering flow cell for TIRFM.

Similar content being viewed by others

References

  1. Bianco, P.R. et al. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules. Nature 409, 374–378 (2001).

    Article  CAS  Google Scholar 

  2. Perkins, T.T. et al. Forward and reverse motion of single RecBCD molecules on DNA. Biophys. J. 86, 1640–1648 (2004).

    Article  CAS  Google Scholar 

  3. Handa, N., Bianco, P.R., Baskin, R.J. & Kowalczykowski, S. Direct visualization of RecBCD movement reveals cotranslocation of the RecD motor after χ recognition. Mol. Cell 17, 745–750 (2005).

    Article  CAS  Google Scholar 

  4. Spies, M. et al. A molecular throttle: the recombination hotspot chi controls DNA translocation by the RecBCD helicase. Cell 114, 647–654 (2003).

    Article  CAS  Google Scholar 

  5. Chemla, Y.R. et al. Mechanism of force generation of a viral DNA packaging motor. Cell 122, 683–692 (2005).

    Article  CAS  Google Scholar 

  6. Grayson, P., Han, L., Winther, T. & Phillips, R. Real-time observations of single bacteriophage lambda DNA ejections in vitro. Proc. Natl. Acad. Sci. USA 104, 14652–14657 (2007).

    Article  CAS  Google Scholar 

  7. Amitani, I., Baskin, R.J. & Kowalczykowski, S.C. Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Mol. Cell 23, 143–148 (2006).

    Article  CAS  Google Scholar 

  8. Bianco, P.R., Bradfield, J.J., Castanza, L.R. & Donnelly, A.N. Rad54 oligomers translocate and cross-bridge double-stranded DNA to stimulate synapsis. J. Mol. Biol. 374, 618–640 (2007).

    Article  CAS  Google Scholar 

  9. van Oijen, A.M. et al. Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder. Science 301, 1235–1238 (2003).

    Article  CAS  Google Scholar 

  10. Wuite, G.J.L., Smith, S.B., Young, M., Keller, D. & Bustamante, C. Single-molecule studies of the effect of template tension on T7 DNA polymerase activity. Nature 404, 103–106 (2000).

    Article  CAS  Google Scholar 

  11. Luo, G., Wang, M., Konigsberg, W.H. & Xie, X.S. Single-molecule and ensemble fluorescence assays for a functionally important conformational change in T7 DNA polymerase. Proc. Natl. Acad. Sci. USA 104, 12610–12615 (2007).

    Article  CAS  Google Scholar 

  12. Tanner, N.A. et al. Single-molecule studies of fork dynamics in Escherichia coli DNA replication. Nat. Struct. Mol. Biol. 15, 170–176 (2008).

    Article  CAS  Google Scholar 

  13. Harada, Y. et al. Single-molecule imaging of RNA polymerase-DNA interactions in real time. Biophys. J. 76, 709–715 (1999).

    Article  CAS  Google Scholar 

  14. Davenport, R.J., Wuite, G.J., Landick, R. & Bustamante, C. Single-molecule study of transcriptional pausing and arrest by E. coli RNA polymerase. Science 287, 2497–2500 (2000).

    Article  CAS  Google Scholar 

  15. Cheng, W., Dumont, S., Tinoco, I. Jr. & Bustamante, C. NS3 helicase actively separates RNA strands and senses sequence barriers ahead of the opening fork. Proc. Natl. Acad. Sci. USA 104, 13954–13959 (2007).

    Article  CAS  Google Scholar 

  16. Dumont, S. et al. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439, 105–108 (2006).

    Article  CAS  Google Scholar 

  17. Shaqfeh, E.S. The dynamics of single-molecule DNA in flow. J. Non-Newt. Fluid Mech. 130, 1–28 (2005).

    Article  CAS  Google Scholar 

  18. Bustamante, C. Unfolding single RNA molecules: bridging the gap between equilibrium and non-equilibrium statistical thermodynamics. Q. Rev. Biophys. 38, 291–301 (2005).

    Article  CAS  Google Scholar 

  19. Brewer, L.R., Corzett, M. & Balhorn, R. Protamine-induced condensation and decondensation of the same DNA molecule. Science 286, 120–123 (1999).

    Article  CAS  Google Scholar 

  20. Bennink, M.L. et al. Single-molecule manipulation of double-stranded DNA using optical tweezers: interaction studies of DNA with RecA and YOYO-1. Cytometry 36, 200–208 (1999).

    Article  CAS  Google Scholar 

  21. Bennink, M.L. et al. Unfolding individual nucleosomes by stretching single chromatin fibers with optical tweezers. Nat. Struct. Biol. 8, 606–610 (2001).

    Article  CAS  Google Scholar 

  22. Bustamante, C., Bryant, Z. & Smith, S.B. Ten years of tension: single-molecule DNA mechanics. Nature 421, 423–427 (2003).

    Article  Google Scholar 

  23. Greene, E.C. & Mizuuchi, K. Direct observation of single MuB polymers: evidence for a DNA-dependent conformational change for generating an active target complex. Mol. Cell 9, 1079–1089 (2002).

    Article  CAS  Google Scholar 

  24. Dame, R.T., Noom, M.C. & Wuite, G.J. Bacterial chromatin organization by H-NS protein unravelled using dual DNA manipulation. Nature 444, 387–390 (2006).

    Article  CAS  Google Scholar 

  25. Galletto, R., Amitani, I., Baskin, R.J. & Kowalczykowski, S.C. Direct observation of individual RecA filaments assembling on single DNA molecules. Nature 443, 875–878 (2006).

    Article  CAS  Google Scholar 

  26. Tan, X., Mizuuchi, M. & Mizuuchi, K. DNA transposition target immunity and the determinants of the MuB distribution patterns on DNA. Proc. Natl. Acad. Sci. USA 104, 13925–13929 (2007).

    Article  CAS  Google Scholar 

  27. Squires, T.M. & Quake, S.R. Microfluidics: fluid physics at the nanoliter scale. Rev. Mod. Phys. 77, 977–1026 (2005).

    Article  CAS  Google Scholar 

  28. Weibel, D.B. & Whitesides, G.M. Applications of microfluidics in chemical biology. Curr. Opin. Chem. Biol. 10, 584–591 (2006).

    Article  CAS  Google Scholar 

  29. Levin, S. & Tawil, G. Effect of surfactants on the diffusion coefficients of proteins, measured by analytical SPLITT fractionation (ASF) in the diffusion mode. J. Pharm. Biomed. Anal. 12, 499–507 (1994).

    Article  CAS  Google Scholar 

  30. Fuh, C.B., Levin, S. & Giddings, J.C. Rapid diffusion coefficient measurements using analytical SPLITT fractionation: application to proteins. Anal. Biochem. 208, 80–87 (1993).

    Article  CAS  Google Scholar 

  31. Ismagilov, R.F., Stroock, A.D., Kenis, P.J.A., Whitesides, G.M. & Stone, H.A. Experimental and theoretical scaling laws for transverse diffusive broadening in two-phase laminar flows in microchannels. Appl. Phys. Lett. 76, 2376–2378 (2000).

    Article  CAS  Google Scholar 

  32. Kamholz, A.E. & Yager, P. Theoretical analysis of molecular diffusion in pressure-driven laminar flow in microfluidic channels. Biophys. J. 80, 155–160 (2001).

    Article  CAS  Google Scholar 

  33. Kamholz, A.E., Schilling, E.A. & Yager, P. Optical measurement of transverse molecular diffusion in a microchannel. Biophys. J. 80, 1967–1972 (2001).

    Article  CAS  Google Scholar 

  34. Lima, R., Wada, S., Takeda, M., Tsubota, K. & Yamaguchi, T. In vitro confocal micro-PIV measurements of blood flow in a square microchannel: the effect of the haematocrit on instantaneous velocity profiles. J. Biomech. 40, 2752–2757 (2007).

    Article  Google Scholar 

  35. Hong, J.W. & Quake, S.R. Integrated nanoliter systems. Nat. Biotechnol. 21, 1179–1183 (2003).

    Article  CAS  Google Scholar 

  36. Psaltis, D., Quake, S.R. & Yang, C. Developing optofluidic technology through the fusion of microfluidics and optics. Nature 442, 381–386 (2006).

    Article  CAS  Google Scholar 

  37. Weibel, D.B., Diluzio, W.R. & Whitesides, G.M. Microfabrication meets microbiology. Nat. Rev. Microbiol. 5, 209–218 (2007).

    Article  CAS  Google Scholar 

  38. Wabuyele, M.B., Ford, S.M., Stryjewski, W., Barrow, J. & Soper, S.A. Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices. Electrophoresis 22, 3939–3948 (2001).

    Article  CAS  Google Scholar 

  39. McDonald, J.C. & Whitesides, G.M. Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. Acc. Chem. Res. 35, 491–499 (2002).

    Article  CAS  Google Scholar 

  40. Qi, S. et al. Microfluidic devices fabricated in poly(methyl methacrylate) using hot-embossing with integrated sampling capillary and fiber optics for fluorescence detection. Lab Chip 2, 88–95 (2002).

    Article  CAS  Google Scholar 

  41. Ladoux, B., Quivy, J.P., Doyle, P.S., Almouzni, G. & Viovy, J.L. Direct imaging of single-molecules: from dynamics of a single DNA chain to the study of complex DNA-protein interactions. Sci. Prog. 84, 267–290 (2001).

    Article  CAS  Google Scholar 

  42. Merenda, F. et al. Refractive multiple optical tweezers for parallel biochemical analysis in micro-fluidics. in Proc. SPIE, 6483, (eds. Andrews, D.L., Galves, E.J. & Nienhuis, G.) 64830A (SPIE, Bellingham, Washington, USA, 2007).

    Google Scholar 

  43. Quake, S.R. & Scherer, A. From micro- to nanofabrication with soft materials. Science 290, 1536–1540 (2000).

    Article  CAS  Google Scholar 

  44. De Jong, J., Lammertink, R.G. & Wessling, M. Membranes and microfluidics: a review. Lab Chip 6, 1125–1139 (2006).

    Article  CAS  Google Scholar 

  45. Ng, J.M., Gitlin, I., Stroock, A.D. & Whitesides, G.M. Components for integrated poly(dimethylsiloxane) microfluidic systems. Electrophoresis 23, 3461–3473 (2002).

    Article  CAS  Google Scholar 

  46. Wuite, G.J.L., Davenport, R.J., Rappaport, A. & Bustamante, C. An integrated laser trap/flow control video microscope for the study of single biomolecules. Biophys. J. 79, 1155–1167 (2000).

    Article  CAS  Google Scholar 

  47. Noom, M.C., van den Broek, B., van Mameren, J. & Wuite, G.J.L. Visualizing single DNA-bound proteins using DNA as a scanning probe. Nat. Methods 4, 1031–1036 (2007).

    Article  CAS  Google Scholar 

  48. Mijatovic, D., Eijkel, J.C. & van den Berg, A. Technologies for nanofluidic systems: top-down vs. bottom-up—a review. Lab Chip 5, 492–500 (2005).

    Article  CAS  Google Scholar 

  49. Rusu, C. et al. Direct integration of micromachined pipettes in a flow channel for single DNA molecule study by optical tweezers. J. Microelectromech. Syst. 10, 238–244 (2001).

    Article  Google Scholar 

  50. Takayama, S. et al. Patterning cells and their environments using multiple laminar fluid flows in capillary networks. Proc. Natl. Acad. Sci. USA 96, 5545–5548 (1999).

    Article  CAS  Google Scholar 

  51. Kenis, P.J. et al. Fabrication inside microchannels using fluid flow. Acc. Chem. Res. 33, 841–847 (2000).

    Article  CAS  Google Scholar 

  52. Sia, S.K. & Whitesides, G.M. Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis 24, 3563–3576 (2003).

    Article  CAS  Google Scholar 

  53. Kim, S., Blainey, P.C., Schroeder, C.M. & Xie, X.S. Multiplexed single-molecule assay for enzymatic activity on flow-stretched DNA. Nat. Methods 4, 397–399 (2007).

    Article  CAS  Google Scholar 

  54. Tanaka, H., Ishijima, A., Honda, M., Saito, K. & Yanagida, T. Orientation dependence of displacements by a single one-headed myosin relative to the actin filament. Biophys. J. 75, 1886–1894 (1998).

    Article  CAS  Google Scholar 

  55. Leuba, S.H., Bennink, M.L. & Zlatanova, J. Single-molecule analysis of chromatin. Methods Enzymol. 376, 73–105 (2004).

    Article  CAS  Google Scholar 

  56. Zheng, H., Tomschik, M., Zlatanova, J. & Leuba, S.H. Evanescent field fluorescence microscopy for analysis of protein/DNA interactions at the single-molecule level. in Protein Protein Interactions, a Molecular Cloning Manual. (eds. Golemis, E. & Adams, P.) 429–444 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2005).

    Google Scholar 

  57. Lee, J.B. et al. DNA primase acts as a molecular brake in DNA replication. Nature 439, 621–624 (2006).

    Article  CAS  Google Scholar 

  58. Brewer, L., Corzett, M. & Balhorn, R. Condensation of DNA by spermatid basic nuclear proteins. J. Biol. Chem. 277, 38895–38900 (2002).

    Article  CAS  Google Scholar 

  59. Brewer, L., Corzett, M., Lau, E.Y. & Balhorn, R. Dynamics of protamine 1 binding to single DNA molecules. J. Biol. Chem. 278, 42403–42408 (2003).

    Article  CAS  Google Scholar 

  60. Moffitt, J.R., Chemla, Y.R., Izhaky, D. & Bustamante, C. Differential detection of dual traps improves the spatial resolution of optical tweezers. Proc. Natl. Acad. Sci. USA 103, 9006–9011 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank C. Bustamante, Kiyoshi Mizuuchi, G. Wuite, H. Gao and M. Gao for critical reading of the manuscript. We would also like to thank K. Mizuuchi and G. Wuite for sharing unpublished flow cell parameters and flow velocities. Funding for the experimental work in the Brewer group was provided by US National Institutes of Health grant HD01387 to L.R.B. Funding for the experimental work in the Bianco group was from US National Institutes of Health grant GM66831 and Susan G. Komen Breast Cancer Foundation grant BCTR0601350 to P.R.B.

Author information

Authors and Affiliations

  1. School of Chemical and Bio-Engineering, Center for Reproductive Biology, Washington State University, PO Box 642710, Pullman, 99164, Washington, USA

    Laurence R Brewer

  2. Department of Microbiology and Immunology and Department of Biochemistry, Center for Single Molecule Biophysics, University at Buffalo, SUNY, Buffalo, 14214, New York, USA

    Piero R Bianco

Authors
  1. Laurence R Brewer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Piero R Bianco
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Laurence R Brewer or Piero R Bianco.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–2, Supplementary Table 1 (PDF 258 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Brewer, L., Bianco, P. Laminar flow cells for single-molecule studies of DNA-protein interactions. Nat Methods 5, 517–525 (2008). https://doi.org/10.1038/nmeth.1217

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1217

This article is cited by

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