Protein-DNA Mapping using an AFM
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Using AFM for mapping protein-DNA interactions
Anthony SalvagnoLaura Pawlikowski
John MontoyaKen Seal
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Outline
• Background• AFM functionalization• Fabrication of device • Applications of device• Conclusions
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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 DNAFrom Wikipedia: Crystal Structureof a protein bound to DNA
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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
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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.
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AFM and Nanofluidics
• AFM has also been incorporated with nanofluidic applications
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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
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AFM Chip
Nano Channel Chip
Chip Design
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Basics of Photolithography
Spin-on Photoresist
Expose Photoresist
Develop Photoresist
Modify Wafer Surface
Anisotropic Etch
Deposit Metal
Selective Plane Etch
Remove Photoresist
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AFM FabricationStep 1: Deep Anisotropic Etch
Step 2: Shallow Anisotropic Etch
Step 3: Silicon Plane Etch
Step 4: Deposit SU-8 PR
Step 5: Deposit Gold Contact
Step 1: Deep Anisotropic Etch
Step 2: Deposit SiO2
Step 3: Deposit Gold Contacts
Step 4: Deposit Indium Bond Metal
Flip-chip Bond Wafer 1 & 2
Silicon Release Wet Etch
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Step 1: Deposit PMMA PR
Step 3: Define Nano Channel Cover
Step 2: Template PMMA PR
Nano Channel FabricationStep 4: Etch Hole
Step 5: Deposit Gold Contacts
Step 6: Deposit Indium Bond Contacts
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Fabricated Device
AFM Chip
Nano Channel Chip
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Device Fabrication
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Device Applications
• Rapid Detection of Protein bound DNA• Useful for:– Gene Expression• Turning genes on / off
– DNA Restriction– DNA Repair– DNA Mutations
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Process
• Loading the chip– Insert digested DNA / Protein / Buffer Mixture
• Capillary Action Feeds the mixture to the AFM tip
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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
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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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Questions?
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