%A Arezi,Bahram %A McKinney,Nancy %A Hansen,Connie %A Cayouette,Michelle %A Fox,Jeffrey %A Chen,Keith %A Lapira,Jennifer %A Hamilton,Sarah %A Hogrefe,Holly %D 2014 %J Frontiers in Microbiology %C %F %G English %K fast PCR,fast cycling,Taq mutants,blood resistant,inhibitor resistant,inhibitor resistant Taq,blood resistant Taq,fast taq mutants %Q %R 10.3389/fmicb.2014.00408 %W %L %M %P %7 %8 2014-August-14 %9 Original Research %+ Bahram Arezi,bahram.arezi@agilent.com %# %! Fast-cycling and inhibitor resistant Taq mutants %* %< %T Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance %U https://www.frontiersin.org/articles/10.3389/fmicb.2014.00408 %V 5 %0 JOURNAL ARTICLE %@ 1664-302X %X 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.