Develop a PCR Protocol for Preparing “sticky end” DNA Handles for Single Molecule Force Spectroscopy Measurements | NSF REU in Integrated Nanomanufacturing

Single molecule force spectroscopy measurements using optical tweezers (OT) allow for insights into the molecular mechanisms of biological processes. In our lab and in collaboration with the lab of Adrian Whitty, we use OT techniques to study the structure and dynamics of the protein, NF-kappa-B essential modulator (NEMO), whose misregulation has a role in many human diseases. As is common in the field, we use extended polymer handles, made out of DNA, to attach the protein to the microscopic beads, which are required for OT measurements. However, because of the unique structure of NEMO and how it must be immobilized for experimentation, we require custom handles that contain single stranded DNA overhangs on one end. The protocol for creating such structures has been published by others, but is not trivially extended to the DNA sequences we require. In this project, Shanen Arellano will adapt the protocol to generate DNA handles with sticky overhangs for single molecule measurements of

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