DNA polymerases are a superfamily of enzymes synthesizing DNA using DNA as a template. They are essential for nucleic acid metabolism and for DNA replication and repair. Modern biotechnology and molecular diagnostics rely heavily on DNA polymerases in analyzing nucleic acids. Among a variety of discovered DNA polymerases, Bst polymerase, a large fragment of DNA polymerase I from Geobacillus stearothermophilus, is one of the most commonly used but is not as well studied as Taq polymerase. The ability of Bst polymerase to displace an upstream DNA strand during synthesis, coupled with its moderate thermal stability, has provided the basis for several isothermal DNA amplification methods, including LAMP, WGA, RCA, and many others. Bst polymerase is one of the key components defining the robustness and analytical characteristics of diagnostic test systems based on isothermal amplification. Here, we present an overview of the biochemical and structural features of Bst polymerase and provide information on its mutated analogs.
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http://dx.doi.org/10.1016/j.csbj.2023.09.008 | DOI Listing |
Biotechnol J
January 2025
School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Loop-mediated isothermal amplification (LAMP) is a detection method widely used in pathogen detection and clinical diagnosis. Nevertheless, it is highly constrained by thermal stability, catalytic activity, and resistance to inhibitors of Bst DNA polymerase. In this study, a novel DNA polymerase was characterized from Clostridium thermocellum, exhibiting potential in LAMP detection.
View Article and Find Full Text PDFFront Microbiol
November 2024
Research Center of Molecular Diagnostics and Sequencing, Research Institute of Tsinghua University in Shenzhen, Shenzhen, China.
Sci Rep
October 2024
Faculty of Science, Department of Molecular Biology and Genetics, Istanbul University, Vezneciler, 34134, Istanbul, Turkey.
Loop-Mediated Isothermal Amplification (LAMP) represents a valuable technique for DNA/RNA detection, known for its exceptional sensitivity, specificity, speed, accuracy, and affordability. This study focused on optimizing a LAMP-based method to detect early signs of Plasmopara halstedii, the casual pathogen of sunflower downy mildew, a severe threat to sunflower crops. Specifically, a set of six LAMP primers (two outer, two inner, and two loop) were designed from P.
View Article and Find Full Text PDFbioRxiv
July 2024
Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL, USA.
Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report new nCas9-DNA polymerase fusion proteins to create chimeric oligonucleotide-directed editing (CODE) systems for search-and-replace genome editing. Through successive rounds of engineering, we developed CODEMax and CODEMax(exo+) editors that achieve efficient genome modifications in human cells with low unintended edits.
View Article and Find Full Text PDFFront Bioeng Biotechnol
July 2024
Research Center of Molecular Diagnostics and Sequencing, Research Institute of Tsinghua University in Shenzhen, Shenzhen, China.
Unveiling the potential application of psychrophilic polymerases as candidates for polymerase-nanopore long-read sequencing presents a departure from conventional choices such as thermophilic stearothermophilus (Bst) renowned for its limitation in temperature and mesophilic phage (phi29) polymerases for limitations in strong exonuclease activity and weak salt tolerance. Exploiting the PB-Bst fusion DNA polymerases from Psychrobacillus (PB) and stearothermophilus (Bst), our structural and biochemical analysis reveal a remarkable enhancement in salt tolerance and a concurrent reduction in exonuclease activity, achieved through targeted substitution of a pivotal functional domain. The sulfolobus 7-kDa protein (Sso7d) emerges as a standout fusion domain, imparting significant improvements in PB-Bst processivity.
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