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Single-molecule DNA nanomanipulation: Improved resolution through use of shorter DNA fragments

Nature Methods volume 2, pages 127–138 (2005)Cite this article

  • Anti-digoxigenin, sheep polyclonal (Roche)

  • Biotin-16-deoxyuridine triphosphate (dUTP) (1 mM; Roche)

  • BSA, fraction V (Roche; stock is 50 mg/ml in water)

  • Digoxigenin-11-dUTP, alkali stable (1 mM; Roche)

  • Magnetic beads, 0.8-μm diameter (4 mg/ml) (New England Biolabs; NEB)

  • Phosphate-buffered saline (PBS; Fluka)

  • Polyglutamic acid, Mw 1,500–3,000 (stock is 10 mg/ml in PBS; Sigma)

  • Polystyrene, Mw ∼280,000 (Sigma; stock is 0.1% in toluene)

  • Plasmid DNA for amplification (Fig. 2)

    Figure 2: Preparation of DNA fragment constructs.
    figure 2

    (a,b) Amplification by PCR is used to prepare a 1-kb multiply biotin-labeled DNA fragment, a 1-kb multiply digoxigenin-labeled DNA fragment and a 2-kb G/C-rich DNA fragment containing the bacteriophage T5 N25 promoter. (c,d) After restriction endonuclease digestion, the 1-kb biotin- and digoxigenin-labeled DNA fragments are ligated to the 2-kb promoter DNA fragment. The resulting DNA fragment can be bound to a streptavidin-coated magnetic bead, and the resulting bead-DNA assembly can be attached to a capillary floor coated with anti-digoxigenin.

    Full size image

  • Primers for amplification (Table 1; Fig. 2)

    Table 1 Preparation of DNA fragment. PCR primers.
    Full size table

  • QIAquick PCR Purification and Gel Extraction kits (Qiagen)

  • Restriction enzymes: MluI and NotI (NEB)

  • RNAP holoenzyme, σ-saturated (Epicentre)

  • Standard buffer (SB): 10 mM potassium phosphate buffer (pH 8), 0.1 mg/ml BSA, 0.1% Tween-20, 10 mM EDTA, 10 mM β-mercaptoethanol

  • T4 DNA ligase (10 U/μl) and 10× reaction buffer (NEB)

  • Thermostable DNA polymerase, high fidelity (3 U/μl; Roche)

  • dNTP mix (2.5 mM each; Roche)

  • Tosyl-activated beads, 3-μm diameter (10 mg/ml; Dynal)

  • Transcription buffer: 25 mM HEPES-NaCl pH 7.5, 75–150 mM NaCl, 10 mM MgCl2, 10 mM β-mercaptoethanol, 0.1 mg/ml BSA, 0.1% Tween-20

Anti-digoxigenin, sheep polyclonal (Roche)

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Figure 1: Experimental approach.
Figure 2: Preparation of DNA fragment constructs.
Figure 3: Detection of RNAP-dependent promoter unwinding: improved resolution through use of shorter DNA fragment.

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Acknowledgements

We thank S. Adhya for plasmid samples. This work was supported by funds from the Institut Jacques Monod, an Action Thématique et Incitative sur Programme grant from the Centre National de la Recherche Scientifique, the Universities of Paris VI and Paris VII, the Fondation pour la Recherche Médicale, and a Cold Spring Harbor Laboratory Fellowship to T.R.S., and by National Institutes of Health grant GM41376 and a Howard Hughes Medical Institute Investigatorship to R.H.E.

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Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, 11724, New York, USA

    Andrey Revyakin & Terence R Strick

  2. Waksman Institute and Department of Chemistry, Howard Hughes Medical Institute, Rutgers University, Piscataway, 08854, New Jersey, USA

    Andrey Revyakin & Richard H Ebright

  3. Institut Jacques Monod, Centre National de la Recherche Scientifique UMR7592, Universités de Paris VI et VII, 2 Place Jussieu, Paris, 75251, Cedex 05, France

    Terence R Strick

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  1. Andrey Revyakin
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  2. Richard H Ebright
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Correspondence to Richard H Ebright or Terence R Strick.

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Revyakin, A., Ebright, R. & Strick, T. Single-molecule DNA nanomanipulation: Improved resolution through use of shorter DNA fragments. Nat Methods 2, 127–138 (2005). https://doi.org/10.1038/nmeth0205-127

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