Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance
| Autor: | Connie J. Hansen, Nancy McKinney, Keith Chen, Holly H. Hogrefe, Jennifer Lapira, Sarah Hamilton, Bahram Arezi, Jeffrey D. Fox, Michelle Cayouette |
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| Rok vydání: | 2014 |
| Předmět: |
blood resistant
inhibitor resistant Taq Microbiology (medical) Genetics inhibitor resistant Mutagenesis Mutant lcsh:QR1-502 Fast pcr Biology Microbiology Molecular biology Phenotype lcsh:Microbiology fast PCR Self-replication fast cycling Original Research Article Ligation Taq mutants Whole blood Taq DNA Polymerase |
| Zdroj: | Frontiers in Microbiology, Vol 5 (2014) Frontiers in Microbiology |
| ISSN: | 1664-302X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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