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DNA tethering

DNA tethering. BFY 2012 Philadelphia. Tethering DNA. Biotin molecule (covalently attached) Lambda phage DNA (48,502 bp = 16.2 m) Digoxigenin (covalently attached). DNA tethering. Polystyrene microsphere (d= 0.8 m), functionalized with streptavidin Lambda phage DNA

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DNA tethering

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  1. DNA tethering BFY 2012 Philadelphia

  2. Tethering DNA Biotin molecule (covalently attached) Lambda phage DNA (48,502 bp= 16.2 m) Digoxigenin (covalently attached)

  3. DNA tethering Polystyrene microsphere (d= 0.8 m), functionalized with streptavidin Lambda phage DNA (48,502 bp= 16.2 m) Glass surface (functionalized with anti-digoxigenin)

  4. Tethered particle motion Random motion of small particle restrained by tether to surface. Can measure distribution of particle positions and determine some material properties of tether (things like tether length and “persistence length”) Only simple “flow cell” needed (no flow). Data collection & analysis straightforward.

  5. Persistence length Comes from “directional correlation function” Can show for our DNAs that

  6. Tethered particle motion exp’t 1. Tether beads & image in microscope with 100x oil immersion objective. 2. Take video (low frame rate, ~10 minutes). Use particle tracking to reduce data to x, y coordinates of beads. Fit histograms of positions to Gaussians and determine sigma.

  7. DNA force extension behavior Use fluid drag force to stretch out DNA. Can measure extension of DNA versus drag force.

  8. DNA force extension behavior Need flow. Need flow cell with tubes attached and a pump (syringe pump).

  9. DNA force extension behavior In practice, need to use magnetic beads and magnet to keep beads from sticking to surface (otherwise, measurement can be very painful…) Calculation of bead positions vs. magnet strength and flow rate is interesting numerical problem (no general analytic solution). Data points were measured by a student. Curves are calculated from script for different assumed magnet force strengths. Student used these results to “calibrate” magnet force at 1 pN.

  10. Biological experiments Enzymes which convert dsDNA to ssDNA (or vice versa) will change extension of DNA and result in bead motion.

  11. Biological experiments DNA polymerase shown. As it replicates the “leading strand,” the “lagging strand” is not replicated and remains ssDNA, and coils up as it leaves the polymerase.

  12. Biological experiments Replication of DNA by the DNA polymerase from 29 bacteriophage measured by students (three different experiments aggregated)

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