Research Project
9558847
Problems and limitations in PCR-based clinical tests can be related to specificity, sensitivity, speed, and presence of inhibitors. Our purpose is to provide point-of-care (POC) devise makers, diagnostic companies, and researchers with novel, specially engineered DNA polymerases to meet the increasing need for reliable, fast, and highly specific PCR, which in addition can tolerate various PCR inhibitors. Such polymerases are highly relevant to the demand for advanced, fast PCR cyclers and POC devices where enzyme speed is a limiting factor in biomarker and pathogen testing. Additionally, the ?hot-start? performance and optimized buffer / additives are important for specificity and reliability. Tolerance to PCR inhibitors is important because inhibitors can cause false negatives, requiring DNA extraction which slows processing, adds cost, may lead to losses of target DNA or RNA, and can contribute to cross-contamination. The performance of the new enzymes will be evaluated in collaboration with highly specialized teams in microfluidic systems for ultrafast PCR devices: the James Landers Lab at the University of Virginia and FluxErgy LLC. The research strategy is to generate novel, in vitro-evolved genetic variants of Taq DNA polymerase, designed for direct PCR detection of various clinically important pathogens and biomarkers. The new enzymes will combine three important qualities: inhibition-resistance (IR), high elongation rate (HER), and specificity / hot start (HS) performance. In addition, some of them will possess reverse transcriptase (RT) activity, thus amplifying both DNA and RNA targets. To this end, we will select fast mutant enzymes by progressively shortening the extension time in our newly developed and highly efficient screening process applied to randomly mutagenized libraries of two inhibition-resistant mutants of Taq, C-66 and E4, recently developed in our labs. E4 is one of our bi-functional enzymes with RT activity. For HS performance, two cold-sensitive mutations, previously characterized by us, will be built-in to the starting mutant clones. Double selection criteria, such as high speed plus hot-start, will also be applied with the use of specially designed ?dimers-prone? primers. Highly purified enzymes of the best mutant candidates will be tri-fold compared to the existing top commercial products for high speed, hot-start, and tolerance to PCR inhibitors. Reaction buffers and PCR enhancer additives will be optimized. The novel enzymes, implemented in microfluidic POC PCR devices, should improve the quality of the current clinical / diagnostics tests in terms of reliability, sampling-to-results time, and specificity, along with reduced false negatives, and cost.
