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.
Highlights
Taq Dissociation constant Kd (DNA) polymerase is still considered the workhorse of PCR, providing great economy and reliability in routine amplification of genomic targets up to 2 kb
Taq mutant libraries were subject to Compartmentalized Self Replication (CSR) selection using progressively shorter extension times, ranging from 1 min down to 6 s per kb of taq pol I
From a screen of several hundred clones, we recovered 24 His-tagged Taq mutants that consistently produce earlier Cq values compared to wild-type Taq in real-time PCR under fast cycling conditions
Summary
Taq DNA polymerase is still considered the workhorse of PCR, providing great economy and reliability in routine amplification of genomic targets up to 2 kb. With an extension rate of 60 nt/s, wild-type Taq produces high amplicon yields after 30–40 cycles using 1 min anneal-extension times. Run times are 1.5–2 h on conventional Peltier-based PCR instruments and approximately 1 h using advanced qPCR instrumentation with improved thermal ramp rates (2.2–3◦C/s). Demand for higher throughput and shorter turn-around-time continues to fuel interest in developing faster PCR instrumentation, along with polymerases with improved kinetic properties. Microchip-based technologies are expected to provide drastically reduced run times (
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have