title: 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.
authors.affiliation: Agilent Technologies La Jolla, CA, USA.
authors.forename: Bahram
authors.initials: B
authors.lastname: Arezi
authors.affiliation: Agilent Technologies La Jolla, CA, USA.
authors.forename: Nancy
authors.initials: N
authors.lastname: McKinney
authors.affiliation: Agilent Technologies La Jolla, CA, USA.
authors.forename: Connie
authors.initials: C
authors.lastname: Hansen
authors.affiliation: Agilent Technologies La Jolla, CA, USA.
authors.forename: Michelle
authors.initials: M
authors.lastname: Cayouette
authors.affiliation: Agilent Technologies La Jolla, CA, USA.
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Fox
authors.affiliation: Agilent Technologies La Jolla, CA, USA.
authors.forename: Keith
authors.initials: K
authors.lastname: Chen
authors.affiliation: Agilent Technologies La Jolla, CA, USA.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lapira
authors.affiliation: Agilent Technologies La Jolla, CA, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Hamilton
authors.affiliation: Agilent Technologies La Jolla, CA, USA.
authors.forename: Holly
authors.initials: H
authors.lastname: Hogrefe
keywords: Taq mutants; blood resistant; fast PCR; fast cycling; inhibitor resistant