| Autor: |
Bahram eArezi, Nancy eMckinney, Connie eHansen, Michelle eCayouette, Jeffrey eFox, Keith eChen, Jennifer eLapira, Sarah eHamilton, Holly eHogrefe |
| Jazyk: |
angličtina |
| Rok vydání: |
2014 |
| Předmět: |
|
| Zdroj: |
Frontiers in Microbiology, Vol 5 (2014) |
| Druh dokumentu: |
article |
| ISSN: |
1664-302X |
| DOI: |
10.3389/fmicb.2014.00408 |
| 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: |
Directory of Open Access Journals |
| Externí odkaz: |
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