Directed Evolution of Enzymes for Streamlined and Reliable RT-qPCR and NGS Workflows

Abstract
Reverse transcription remains an essential and sometimes problematic initial step in methods and workflows for the analysis of RNA by NGS or PCR-based amplification methods. Despite advancements in these technologies and the introduction of engineered reverse transcriptases, efficient conversion of RNAs that form stable secondary structures, and/or the presence of inhibitors in sample matrix can influence the efficacy of first-strand synthesis, introducing bias in RNA sequence coverage or transcript enumeration. This talk will describe novel thermostable, inhibitor tolerant RNA directed DNA polymerases obtained through our molecular screening and directed evolution program and their application to streamlined workflows for RT-PCR and NGS methods. Collectively, the properties of these new enzymes and associated reagent systems offer the promise to simplify, accelerate and improve the reliability and flexibility of detection and analysis of mRNA, noncoding RNA and viral targets.