Using Atomic Force Microscopy to Study DNA Structure

1
Using Atomic Force Microscopy to Study DNA
Structure

• Michele Pugh
• Senior Pratt Engineering Undergraduate Fellow
• Mechanical Engineering
• Associate Professor Piotr Marszalek
• March 30, 2005

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Goals

• Can techniques of atomic force microscopy can be
  used to examine DNA structure?
• Observe DNA structure and detect damage to DNA
  molecules using two techniques using an atomic
  force microscope
• Imaging
• Force spectroscopy
• Become proficient in both imaging and force
  spectroscopy (stretching)
• Develop consistent procedures for both techniques

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AFM imaging of DNA

• As the cantilever tip scans the sample surface,
  the microscope produces a topographical image of
  the sample by measuring the deflection of the tip

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Sample preparation for imaging

• Mica surface is treated with APTES and the DNA
  and proteins are diluted with TE buffer and MgCl2
  solution
• The DNA-protein solution heated to normal body
  temperature to induce the proteins to bind to the
  DNA
• DNA solution deposited on the treated mica and
  incubated at room temperature

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Imaging DNA with attached proteins

• DNA in yellow at a higher elevation than the
  mica surface
• Proteins in white at a higher elevation than
  the DNA
• Sample preparation is very important in order to
  minimize the appearance of aggregates and foreign
  particles in the images

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Using force spectroscopy to measure the
elasticity of DNA

• By recording data about the displacement of the
  sample and the deflection of the cantilever tip,
  the length and tension of the DNA molecule can be
  determined
• Elasticity curve of control DNA is different than
  that of DNA damaged by radiation

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Sample preparation for stretching

• A much higher concentration of DNA is needed in
  order to increase the chances that the cantilever
  will pick up a molecule
• DNA diluted in TE buffer with NaCl solution to
  stabilize the molecule
• DNA solution deposited onto pure gold substrate

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Measuring elasticity of control DNA
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What control DNA should look like

• Obtained by Gwangrog Lee

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Measuring elasticity of DNA exposed to gamma
radiation
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Conclusions

• Small mechanical manipulations of DNA molecules
  can be used to study the structure of the
  molecule
• Techniques of atomic force microscopy can be used
  to detect changes in the structure of DNA
  molecules associated with radiation damage

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Possible applications of this research

• Detection of DNA damage induced by environmental
  agents, such as UV or gamma radiation DNA,
  specifically those that cause skin cancer
• Detection of spontaneous mutations in DNA
  molecules
• Better visualization of the DNA and repair
  protein complex

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Acknowledgements

• Dr. Piotr Marszalek
• Dr. Qinming Zhang
• Gwangrog Lee
• Dean Martha Absher and the Pratt Fellows Program