Molecular Evolution for the Development of an Inhibitor-Resistant DNA Polymerase for Improved Target Amplification and Quantification of Crude Samples with Real-Time PCR

As sample quantities continue to grow for high throughput gene expression and genotyping real-time PCR applications such as pathogen detection, mouse genotyping, and GMO screening, so does the need for greater levels of detection and process efficiency. Enzymatic inhibition is an inherent challenge of PCR amplification when working with complex biological samples, thus requiring DNA extraction and purification methods prior to sample analysis. Robust purification methods are available, but impose additional time, cost, and DNA loss with even the most efficient processes that can potentially lead to false negatives of low copy targets. While crude extraction methods alleviate process burdens, higher levels of inhibitors lead to inefficient amplification and variable results.

Utilizing high-throughput molecular evolution, we have developed a novel, highly processive third-generation DNA polymerase not only capable of crude sample amplification, but also with 5’-3’ exonuclease activity required for probe-based qPCR. In this study, we assessed the ability of this enzyme to amplify a variety of blood, tissue, and plant DNA templates after crude sample extraction and in the presence of relevant inhibitors in probe-based assays. We compared the novel polymerase with current industry-leading reagents, focusing on PCR efficiency and threshold cycle (C_q) metrics. Our novel polymerase was capable of amplifying DNA from samples after crude extraction without an observable delay in C_q. At inhibitor concentrations that did not result in a >1 C_q delay for the engineered enzyme, competing reagents typically displayed much greater delays in their corresponding C_q values.