Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance

Abstract

Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.

Combined elevation of TRIB2 and MAP3K1 indicates poor prognosis and chemoresistance to temozolomide in glioblastoma

“…Increasing evidence has suggested that rapid acquired therapeutic resistance is the major cause of the failure of standard comprehensive treatments, leading to the recurrence and high mortality of glioma . Various molecular pathways have been shown to be responsible for the acquisition of therapeutic resistance in glioma . Therefore, it is essential to identify the mechanisms, especially prognostic biomarkers, underlying therapeutic resistance in glioma.…”

“…29,30 Various molecular pathways have been shown to be responsible for the acquisition of therapeutic resistance in glioma. 26,[31][32][33] Therefore, it is essential to identify the mechanisms, especially prognostic biomarkers, underlying therapeutic resistance in glioma. In this study, we analyzed the transcriptome expression profiles of 3 published GEO databases related to pathological classification, radioresistance, and TMZ resistance and identified TRIB2…”

Engineered Protein Machines: Emergent Tools for Synthetic Biology

“…Following heat treatment to degrade the components of the cell, only the polymerase and its template gene remain in the emulsion, and a PCR reaction can be initiated by polymerase variants that are functional under conditions that can be customized to select for numerous desired phenotypes. The CSR scheme has been used to generate novel DNA polymerases with a number of unique features, including increased thermostability, the ability to bypass blocking lesions, increased DNA binding affinity, resistance to inhibitors, and decreased uracil binding (Arezi et al, 2014;Ghadessy et al, 2001Ghadessy et al, , 2004Tubeleviciute and Skirgaila, 2010).…”

“…(3) Either (a) compartmentalized self-replication (CSR) is performed under selective conditions to preferentially amplify the genes of productive DNA polymerases (green circles) rather than unproductive DNA polymerases (red polygons) (Ghadessy et al, 2001) or (b) compartmentalized self-tagging (CST) is performed to tag productive polymerase genes with a biotin-labeled primer before (4) streptavidin precipitation . These schemes have been used to generate new polymerases with increased thermostability, the ability to bypass blocking lesions, increased DNA binding affinity, resistance to inhibitors, decreased uracil binding, and, ultimately, synthesis of unnatural genetic polymers (Arezi et al, 2014;Ghadessy et al, 2001Ghadessy et al, , 2004Tubeleviciute and Skirgaila, 2010). Green bars represent genes for productive polymerase variants; red bars represent genes for unproductive polymerase variants.…”

DNA polymerases in Biotechnology

“…Several papers characterize DNA polymerases that tolerate PCR inhibitors and allow rapid DNA amplification from clinical samples without DNA purification, thereby reducing analysis time, cost, and potential for contamination. The contribution by Killelea et al demonstrates that a Family D polymerase from Pyrococcus abysii is tolerant to high concentrations of PCR inhibitors while Arezi and colleagues describe a method to select for DNA polymerase variants that enable direct PCR from whole blood (Arezi et al, 2014;Killelea et al, 2014).…”