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Engineering psychrophilic polymerase for nanopore long-read sequencing

Front Bioeng Biotechnol. 2024-07; 
Yaping Sun , Danny Hsu Ko , Jie Gao , Kang Fu , Yuanchen Mao , Yun He , Hui Tian
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PCR Cloning and Subcloning Based on this, PB polymerase and PBmutant variants (PB-422, PB-DXE PB-822, PB-732, and PB-Bst) were synthesized and cloned into the Pet21a plasmid by GenScript (China). Phi29 wild-type and its mutant, ZG_O were also synthesized by GenScript. Get A Quote

摘要

Unveiling the potential application of psychrophilic polymerases as candidates for polymerase-nanopore long-read sequencing presents a departure from conventional choices such as thermophilic Bacillus stearothermophilus (Bst) renowned for its limitation in temperature and mesophilic Bacillus subtilis phage (phi29) polymerases for limitations in strong exonuclease activity and weak salt tolerance. Exploiting the PB-Bst fusion DNA polymerases from Psychrobacillus (PB) and Bacillus 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 domai... More

关键词

exonuclease activity; incorporation of polymer-tagged nucleotides; psychrophilic polymerases; salt tolerance; sequencing by synthesis (SBS).