Protein-DNA Mapping using an AFM

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This is the presentation based on our propsal of the same name. The presenters were Laura Pawlikowski, John Montoya, Ken Seal, and myself. We discuss functionalizing an AFM tip with antibodies for protein detection and discuss several possible uses for such a device. Conceptually, proteins bound to DNA can be flowed down a nanochannel and can be detected by an AFM tip in the channel. The detection will be based on the interaction between antibodies and their antigens (the proteins).

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Protein-DNA Mapping using an AFM

  1. 1. Using AFM for mapping protein-DNA interactions<br />Anthony Salvagno<br />Laura Pawlikowski<br />John Montoya<br />Ken Seal<br />
  2. 2. Outline<br />Background<br />AFM functionalization<br />Fabrication of device <br />Applications of device<br />Conclusions<br />
  3. 3. Protein-DNA Interactions<br />Some Examples<br />Restriction Enzymes<br />Cuts DNA to protect of viral infection in bacteria<br />Transcription<br />Lots of proteins bind to control gene expression<br />DNA Replication<br />Proteins unwind DNA and copy DNA<br />From Wikipedia: Crystal Structure<br />of a protein bound to DNA<br />
  4. 4. Gene Expression: One of the many reasons to map PDI<br />DNA Transcription<br />Copies DNA into mRNA<br />RNA Translation<br />Uses mRNA to make Proteins<br />Each step mediated by protein-DNA interactions<br />Mutations change everything<br />Daviddarling.info<br />
  5. 5. AFM and DNA mapping<br />Atomic force microscopy (AFM) has been used in many applications involving DNA<br />mapping exonuclease activities of DNA <br />regular tip in tapping mode. <br />interaction between thalidomide and DNA <br />studied the topography of the substance. <br />
  6. 6. AFM and Nanofluidics<br />AFM has also been incorporated with nanofluidic applications<br />
  7. 7. AFM functionalization<br />How chips have been functionalized<br />UV light used to purify chips<br />Attaching antibodies to chips<br />Chips allowed to sit in solution allowed to incubate, allowing the antibodies to attach <br />Capacitance measurements<br />
  8. 8. Chip Design<br />AFM Chip<br />Nano Channel Chip<br />
  9. 9. Basics of Photolithography<br />Remove Photoresist<br />Modify Wafer Surface<br />Spin-on Photoresist<br />Anisotropic Etch<br />Expose Photoresist<br />Deposit Metal<br />Develop Photoresist<br />Selective Plane Etch<br />
  10. 10. AFM Fabrication<br />Step 1: Deep Anisotropic Etch<br />Step 2: Deposit SiO2<br />Step 1: Deep Anisotropic Etch<br />Step 3: Deposit Gold Contacts<br />Step 2: Shallow Anisotropic Etch<br />Step 4: Deposit Indium Bond Metal<br />Step 3: Silicon Plane Etch<br />Flip-chip Bond Wafer 1 & 2<br />Step 4: Deposit SU-8 PR<br />Step 5: Deposit Gold Contact<br />Silicon Release Wet Etch<br />
  11. 11. Nano Channel Fabrication<br />Step 1: Deposit PMMA PR<br />Step 4: Etch Hole <br />Step 2: Template PMMA PR <br />Step 5: Deposit Gold Contacts <br />Step 3: Define Nano Channel Cover<br />Step 6: Deposit Indium Bond Contacts <br />
  12. 12. Fabricated Device<br />AFM Chip<br />Nano Channel Chip<br />
  13. 13. Device Fabrication<br />
  14. 14. Device Applications<br />Rapid Detection of Protein bound DNA<br />Useful for:<br />Gene Expression<br />Turning genes on / off<br />DNA Restriction<br />DNA Repair<br />DNA Mutations<br />
  15. 15. Process<br />Loading the chip<br />Insert digested DNA / Protein / Buffer Mixture<br />Capillary Action Feeds the mixture to the AFM tip<br />
  16. 16. Problems<br />DNA / Protein mixture must enter in a head to tail fashion.<br />enters sideways<br />folds back on itself<br />flow out of the channel<br />Researcher must know preliminary data regarding the DNA sequence<br />DNA enter 3&apos; or 5&apos; end first<br />Positive / Negative results must be followed up.<br />CHIP<br />
  17. 17. Conclusion<br />The chip offers researchers<br />inexpensive<br />high-throughput<br />rapid Protein bound DNA detection device.<br />Save researchers both time an money. <br />Ability to conduct entire experiment on a chip<br />
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