Template Switching 2.0: A Microfluidic Approach
Amanda Khoo
Faculty Mentor: Ángel Islas
Template switching is a phenomenon in which an enzyme synthesizes DNA across two non-continuous, juxtaposed DNA templates through microhomology between the two strands [1]. Template switching activity and substrate preference have been previously detailed by Garcia et. al using a highly sensitive template switching assay designed in the Islas lab. Continued work using this assay revealed other enzymes that have template-switching activity, including the p66 subunit of HIV reverse transcriptase, further validating the assay’s capability as a standard for studying template switching. This assay, however, is time, labor and reagent intensive. The advent of microfluidics (micro-volume experiments on designed micro-platforms) for biological purposes has opened the doors for increasingly rapid, sensitive biological assays. This project aims to move the existing template switching assay to a micro-platform for rapid, cheaper analysis and ease of experimental execution with comparable sensitivities to the existing template switching assay.
[1] García, Patty B., Nicole L. Robledo, and Ángel L. Islas. “Analysis of Non-Template-Directed Nucleotide Addition and Template Switching by DNA Polymerase.” Biochemistry 43.51: 16515-6524 (2004).