Single DNA molecule grafting and manipulation using a combined atomic force microscope and an optical tweezer

Applied Physics Letters

Volumn 71, Issue 25, 1997, Pages 3727-3729

  Shivashankar, G V ^a   Libchaber, A ^a  

^a ROCKEFELLER UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EID: 0001748585     PISSN: 00036951     EISSN: None     Source Type: Journal    
DOI: 10.1063/1.120495     Document Type: Article

References (17)

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4. The trapping force is calibrated using Stokes drag method by measuring the velocity of the translational stage at which the bead escapes (F = 6 πηaν, where η is the water viscosity, ν the velocity, and a the bead radius). Independently we also calibrate the trapping stiffness by measuring rms fluctuations of the trapped bead. R. M. Simmons, J. T. Finer, S. Chu, and J. Spudich, Biophys. J. 70, 1813 (1996); K. Svoboda and S. M. Block, Annu. Rev. Biophys. Biomol. Struct. 23, 247 (1994).
5. The trapping force is calibrated using Stokes drag method by measuring the velocity of the translational stage at which the bead escapes (F = 6 πηaν, where η is the water viscosity, ν the velocity, and a the bead radius). Independently we also calibrate the trapping stiffness by measuring rms fluctuations of the trapped bead. R. M. Simmons, J. T. Finer, S. Chu, and J. Spudich, Biophys. J. 70, 1813 (1996); K. Svoboda and S. M. Block, Annu. Rev. Biophys. Biomol. Struct. 23, 247 (1994).
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8. 2, where m is the effective mass, and ω the resonance frequency of the cantilever. A more accurate calibration is directly obtained by measuring the rms thermal fluctuations of the cantilever and thereby estimating its stiffness. The accuracy in determining the stiffness of the cantilever, in our case 10 picoN/nm, is known within 20%.
9. Tethered beads are prepared by attaching one end of a double stranded λ DNA polymer (Promega) to a coverslip and the other end to a 3.2 μm latex bead (Polysciences). One end of the DNA is first modified to have a biotinylated molecule on one strand. It is attached to a 3.2 μm latex bead (coated with streptavidin) by making use of the biotin-streptavidin linker. The other end of DNA is then modified to have biotin molecule on the other strand. It is then attached to the coverslip (coated with streptavidin using surface adsorption). The experiments are carried out at pH 7.4 and in 0.1 M phosphate buffered saline. Typically the ratio of the number of beads to DNA molecules ∼1:10, for DNA attachment to the coverslip.
10. The absorption is so strong that the tip temperature can easily reach 100°C, creating a gas bubble in the surrounding fluid. If the laser is focused (optical power in the range 20-60 mW) onto the cantilever for more than a few tens of seconds, a small flow is setup. As the flow converges to the cantilever, the arrival of free untethered beads leads to a perfect close packed assembly of beads around the cantilever. One thus must be careful when using the tweezer close to the cantilever tip.
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15. 10,11 for λ DNA. However the force values are within the error bars of our estimates for the cantilever stiffness. A careful consideration of the magnitude of the overstretching transition and the force curve is beyond the scope of this letter.
16. J. Marko and E. Siggia, Macromolecules 28, 8759 (1995).
17. This work shows the possibility of surface modification, of a cantilever tip for example, by grafting μm size beads (with specific chemical groups or specific DNA sequences attached). It is also possible to optically graft beads with known DNA sequences, in a regular array on a substrate.