Develop a PCR Protocol for Preparing “sticky end” DNA Handles for Single Molecule Force Spectroscopy Measurements

Project Description

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 NEMO. She will then use these constructs to begin studying NEMO using OT methods.

Mentors

Masha Kamenetska, PI  Favian Liu

• Use PCR techniques to create a basic DNA construct that has been made in the lab previously. This construct contains no sticky overhangs, but is functionalized on the ends with biotin and anti-DIG linkers for attaching to microscale beads
• Use the Lumicks OT instrument in the Kamenetska lab to chemically link the DNA to the beads and apply and measure forces on the control DNA from goal 1
• Adapt the “sticky end” PCR protocol from the literature, to generate the DNA handles required for NEMO experiments
• Test the efficacy of the new DNA handles by performing OT measurements
• Learn biochemistry techniques such as PCR and Gel electrophoresis for generating DNA constructs with desired sequence and length
• Learn the basics of optical trapping methodology and data analysis
• Learn to use the Lumicks OT instrument in the Kamenetska lab
• Become introduced to data analysis in python

Timeline