Robust and Fast Taq Mutants Designed for Direct and Rapid Point of Care Tests

Problems and limitations in PCR based clinical tests can be related to specificitysensitivityspeedand presence of inhibitorsOur purpose is to provide point of carePOCdevise makersdiagnostic companiesand researchers with novelspecially engineered DNA polymerases to meet the increasing need for reliablefastand highly specific PCRwhich in addition can tolerate various PCR inhibitorsSuch polymerases are highly relevant to the demand for advancedfast PCR cyclers and POC devices where enzyme speed is a limiting factor in biomarker and pathogen testingAdditionallythehot startperformance and optimized bufferadditives are important for specificity and reliabilityTolerance to PCR inhibitors is important because inhibitors can cause false negativesrequiring DNA extraction which slows processingadds costmay lead to losses of target DNA or RNAand can contribute to cross contaminationThe performance of the new enzymes will be evaluated in collaboration with highly specialized teams in microfluidic systems for ultrafast PCR devicesthe James Landers Lab at the University of Virginia and FluxErgy LLCThe research strategy is to generate novelin vitro evolved genetic variants of Taq DNA polymerasedesigned for direct PCR detection of various clinically important pathogens and biomarkersThe new enzymes will combine three important qualitiesinhibition resistanceIRhigh elongation rateHERand specificityhot startHSperformanceIn additionsome of them will possess reverse transcriptaseRTactivitythus amplifying both DNA and RNA targetsTo this endwe 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 TaqCand Erecently developed in our labsEis one of our bi functional enzymes with RT activityFor HS performancetwo cold sensitive mutationspreviously characterized by uswill be built in to the starting mutant clonesDouble selection criteriasuch as high speed plus hot startwill also be applied with the use of specially designeddimers proneprimersHighly purified enzymes of the best mutant candidates will be tri fold compared to the existing top commercial products for high speedhot startand tolerance to PCR inhibitorsReaction buffers and PCR enhancer additives will be optimizedThe novel enzymesimplemented in microfluidic POC PCR devicesshould improve the quality of the current clinicaldiagnostics tests in terms of reliabilitysampling to results timeand specificityalong with reduced false negativesand cost Project Narrative The project proposes a way of achieving fastermore specific and reliable clinical tests used in diagnostics and management of infectious and genetic diseases with less false negative resultsThe work will implement novel genetically engineered forms of the enzyme that is essential for such testswith high tech point of care portable devicesBesides improved quality of the clinical teststhe project should deliver reduced turnaround time at lower cost