The document discusses using atomic force microscopy (AFM) to map protein-DNA interactions. It outlines how AFM chips can be fabricated through photolithography to include nanochannels. The chips allow rapid detection of protein-bound DNA, which has applications for studying gene expression, DNA restriction, and DNA mutations. Questions from researchers using the technique are also addressed.

1. Using AFM for mapping protein-DNA interactions Anthony Salvagno Laura Pawlikowski John Montoya Ken Seal

2. Outline Background AFM functionalization Fabrication of device Applications of device Conclusions

3. Protein-DNA Interactions Some Examples Restriction Enzymes Cuts DNA to protect of viral infection in bacteria Transcription Lots of proteins bind to control gene expression DNA Replication Proteins unwind DNA and copy DNA From Wikipedia: Crystal Structure of a protein bound to DNA

4. Gene Expression: One of the many reasons to map PDI DNA Transcription Copies DNA into mRNA RNA Translation Uses mRNA to make Proteins Each step mediated by protein-DNA interactions Mutations change everything Daviddarling.info

5. AFM and DNA mapping Atomic force microscopy (AFM) has been used in many applications involving DNA mapping exonuclease activities of DNA regular tip in tapping mode. interaction between thalidomide and DNA studied the topography of the substance.

6. AFM and Nanofluidics AFM has also been incorporated with nanofluidic applications

7. AFM functionalization How chips have been functionalized UV light used to purify chips Attaching antibodies to chips Chips allowed to sit in solution allowed to incubate, allowing the antibodies to attach Capacitance measurements

8. Chip Design AFM Chip Nano Channel Chip

9. Basics of Photolithography Remove Photoresist Modify Wafer Surface Spin-on Photoresist Anisotropic Etch Expose Photoresist Deposit Metal Develop Photoresist Selective Plane Etch

10. AFM Fabrication Step 1: Deep Anisotropic Etch Step 2: Deposit SiO2 Step 1: Deep Anisotropic Etch Step 3: Deposit Gold Contacts Step 2: Shallow Anisotropic Etch Step 4: Deposit Indium Bond Metal Step 3: Silicon Plane Etch Flip-chip Bond Wafer 1 & 2 Step 4: Deposit SU-8 PR Step 5: Deposit Gold Contact Silicon Release Wet Etch

11. Nano Channel Fabrication Step 1: Deposit PMMA PR Step 4: Etch Hole Step 2: Template PMMA PR Step 5: Deposit Gold Contacts Step 3: Define Nano Channel Cover Step 6: Deposit Indium Bond Contacts

12. Fabricated Device AFM Chip Nano Channel Chip

13. Device Fabrication

14. Device Applications Rapid Detection of Protein bound DNA Useful for: Gene Expression Turning genes on / off DNA Restriction DNA Repair DNA Mutations

15. Process Loading the chip Insert digested DNA / Protein / Buffer Mixture Capillary Action Feeds the mixture to the AFM tip

16. Problems DNA / Protein mixture must enter in a head to tail fashion. enters sideways folds back on itself flow out of the channel Researcher must know preliminary data regarding the DNA sequence DNA enter 3' or 5' end first Positive / Negative results must be followed up. CHIP

17. Conclusion The chip offers researchers inexpensive high-throughput rapid Protein bound DNA detection device. Save researchers both time an money. Ability to conduct entire experiment on a chip

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