Abstract
We report the development of a magnetic tweezers that can be used to micromanipulate single DNA molecules by applying picoNewton (pN)-scale forces in the horizontal plane. The resulting force–extension data from our experiments show high-resolution detection of changes in the DNA tether’s extension: ~0.5 pN in the force and <10 nm change in extension. We calibrate our instrument using multiple orthogonal techniques including the well-characterized DNA overstretching transition. We also quantify the repeatability of force and extension measurements, and present data on the behavior of the overstretching transition under varying salt conditions. The design and experimental protocols are described in detail, which should enable straightforward reproduction of the tweezers.
Highlights
In the past 25 years, many new techniques for the micromanipulation of single DNA molecules and DNA-protein complexes have been developed
4 Lo Ko In Equation (2), fz is the applied force, kB is Boltzmann’s constant, T is the absolute temperature of ~297 K, b is the persistence length of 50 nm, Lo is the contour length of 16.4 μm, Ko is the elastic modulus of DNA which is ~1000 pN, and z is the DNA’s thermally-averaged end-to-end extension, which is the distance between the intensity-weighted centroids of the two beads—see Figure 2 caption
The standard errors (SE) in the force are shown as blue lines; we did not include the S.E. of the DNA extension in Figure 2 because their magnitudes are too small compared to the magnitude of the S.E. of the force
Summary
In the past 25 years, many new techniques for the micromanipulation of single DNA molecules and DNA-protein complexes have been developed. Instrument designs that compensate for sample cell drift—mechanical drift can affect extension measurements in long-duration (~1 h) experiments—have been developed [23,24] These advances have added complexity to vertical magnetic tweezers and required sophisticated calibration techniques to determine the relation between diffraction rings and DNA extension. Another option is to generate the forces on DNA tethers along the focal plane [24,25,26,27,28]. We calibrate our force–extension measurements using the DNA overstretching transition
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