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| Magnetic Tweezer SetupFigure 4 shows a magnetic Tweezer setup, which was used to analyse DNA translocation by R.EcoR124I. DNA, with a single site for the EcoR124I enzyme, is surface attached to a layer of anti-digoxygenin (DIG) through incorporation of DIG into the end of the DNA molecules. The other end of the DNA molecule is attached to a streptavidin-coated paramagnetic bead through incorporation of biotin into the DNA. The bead can be suspended in space using external magnets and forces applied to the DNA by lowering or raising the magnets. An inverted microscope can visualise the beads and determine their height above the objective through a simple computer programme that measures the number and spacing of diffraction rings. The z-height is accurate to 10nm or better and can be used to follow the DNA translocation process (Figure 5). We have determined the speed of translocation for both the R1- and R2-complexes (Figures 5 and 6). These studies also allowed us to show the mechanism for re-setting at the end of a translocation event was due to dissociation of the HsdR subunits from the EcoR124I enzyme (Figure 7) (Seidel et al. 2004). |
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© University of
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