ArticleFrontiers in microbiology2014
Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.
Article in Frontiers in microbiology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed.
- Mechanism and application of Taq DNA polymerase in TaqMan qPCR.Frontiers in bioengineering and biotechnology · 2026Article
- Engineering of novel DNA polymerase variants for single enzyme quantitative multiplex reverse transcription-PCR.Scientific reports · 2025Article
- Engineering a DNA polymerase for modifying large RNA at specific positions.Nature chemistry · 2025Article
- Nucleic acid joining enzymes: biological functions and synthetic applications beyond DNA.The Biochemical journal · 2025Review
- Enhancing the reverse transcriptase function in Taq polymerase via AI-driven multiparametric rational design.Frontiers in bioengineering and biotechnology · 2024Article
- Thermally controlled intein splicing of engineered DNA polymerases provides a robust and generalizable solution for accurate and sensitive molecular diagnostics.Nucleic acids research · 2023Article
- Ultrahigh-Throughput Enzyme Engineering and Discovery inChemical reviews · 2023Review
- Modified Taq DNA Polymerase for Allele-Specific Ultra-Sensitive Detection of Genetic Variants.The Journal of molecular diagnostics : JMD · 2022Article
- Loop-Mediated Isothermal Amplification: From Theory to Practice.Russian journal of bioorganic chemistry · 2022Article
- Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey.Angewandte Chemie (International ed. in English) · 2021Review
- Directed Evolution Methods for Enzyme Engineering.Molecules (Basel, Switzerland) · 2021Review
- PCR inhibition in qPCR, dPCR and MPS-mechanisms and solutions.Analytical and bioanalytical chemistry · 2020Review
- Combined elevation of TRIB2 and MAP3K1 indicates poor prognosis and chemoresistance to temozolomide in glioblastoma.CNS neuroscience & therapeutics · 2020Article
- Identification of Thermus aquaticus DNA polymerase variants with increased mismatch discrimination and reverse transcriptase activity from a smart enzyme mutant library.Scientific reports · 2019Article
- Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR.Analytical and bioanalytical chemistry · 2018Article
- Engineering processive DNA polymerases with maximum benefit at minimum cost.Frontiers in microbiology · 2014Article
- DNA polymerases in biotechnology.Frontiers in microbiology · 2014Article
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Authors and funding
9 authors.
Funding
No grant is acknowledged in the PubMed record.
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.
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