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Micro-Imaging of DNA-Artificial Nucleosome Aggregation

Intro. DNA is a negatively charged polymer which folds on itself during compaction. The condensation pathway is 3-D, and not yet fully understood. . Scale of Occurrence. The strands of DNA and special proteins involved are encompassed in a cell nucleus with a diameter of about two millionth of a meter. .

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Micro-Imaging of DNA-Artificial Nucleosome Aggregation

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    1. Micro-Imaging of DNA-Artificial Nucleosome Aggregation Tejdev Sandhu, Rajagopal Krishnan, Thomas Nordlund

    2. Intro DNA is a negatively charged polymer which folds on itself during compaction. The condensation pathway is 3-D, and not yet fully understood.

    3. Scale of Occurrence The strands of DNA and special proteins involved are encompassed in a cell nucleus with a diameter of about two millionth of a meter.

    8. Research Goals Image aggregation-in-action in order to learn more about the process. Overcome previous imaging limitations through chemical manipulation of the solution. Form a proposed charge structure.

    9. Methods in Brief .043 ?m and .93 ?m polystyrene latex spheres were used as artificial nucleosomes at purchased from Interfacial Dynamics Corp. Herring sperm and lambda phage DNA were purchased from Sigma-Aldrich 50 mM Tris buffer (pH ~ 7.3), containing 100 mM KCl and .1 mM EDTA was used to dissolve the DNA

    10. Imaging Conducted with an IX70 inverted system light microscope Images taken with an Olympus F-View CCD camera Images taken at 20, 40, and 60 minute intervals

    11. 20 µM Herring Sperm DNA

    12. 5 µM Herring Sperm DNA - 43 nm Spheres

    13. Lambda Phage DNA Beaded String Aggregates

    14. Glycerol Tests

    15. Fluorescence vs. Bright-Field Imaging

    16. Charge Structure Calculations Determination of average surface area per sphere given surface area per gram, grams per milliliter, and spheres per milliliter Total charge calculation by subtracting net determined carboxyl groups from net determined amino groups

    17. Proposed 43 nm Charge Structure Diagram

    18. General Conclusions Several in vitro images and movies were obtained The DNA process can be controlled to look beyond apparent randomness Staining with YO-YO1 iodide verifies the aggregates contain high concentrations of DNA and are not formed by impurities The rudimentary charge structures would benefit greatly by further experiments to confirm theoretical calculations

    19. Implications Genetic research Pharmaceuticals

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