Polymerase Engineering to Expand Access to Nucleic Acid Diagnostics and Technologies

Abstract

Diagnostic and molecular biology workflows are often complex when first developed, and simplifying them is a key step in expanding their use from high-resource labs to low-resource settings such as clinics and developing countries. While dedicated devices can go about this task by automating steps and increasing throughput, an alternative and powerful approach is to employ dedicated biomolecular devices – proteins and enzymes such as DNA polymerases – to automate and streamline processes at the molecular scale. In this thesis, I describe methods and platforms that exemplify this “biology-first” mindset, its advantages, and its limitations. I cover two main projects that employ this approach. The first project synthesizes existing biomolecular technologies to demonstrate a novel format for HIV viral load testing with drastically reduced requirements for instrumentation and user handling. I show that this novel format can quantify HIV nucleic acids with accuracy comparable to clinically used assays, but does not require a thermal cycler instrument and can be interpreted by cell phone photography or naked eye. The second project demonstrates a platform for high-throughput screening and evolution of DNA polymerases. I propose that this platform can be used to rapidly develop novel polymerases for applications such as direct nucleic acid diagnostics with minimal or no sample preparation.