Using AFM for mapping protein-DNA interactions

Anthony SalvagnoLaura Pawlikowski

John MontoyaKen Seal

Outline

• Background• AFM functionalization• Fabrication of device • Applications of device• Conclusions

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

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

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.

AFM and Nanofluidics

• AFM has also been incorporated with nanofluidic applications

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

AFM Chip

Nano Channel Chip

Chip Design

Basics of Photolithography

Spin-on Photoresist

Expose Photoresist

Develop Photoresist

Modify Wafer Surface

Anisotropic Etch

Deposit Metal

Selective Plane Etch

Remove Photoresist

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

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

Fabricated Device

AFM Chip

Nano Channel Chip

Device Fabrication

Device Applications

• Rapid Detection of Protein bound DNA• Useful for:– Gene Expression• Turning genes on / off

– DNA Restriction– DNA Repair– DNA Mutations

Process

• Loading the chip– Insert digested DNA / Protein / Buffer Mixture

• Capillary Action Feeds the mixture to the AFM tip

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

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

Questions?

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