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Unraveling the salt tolerance of Phi29 DNA polymerase using compartmentalized self-replication and microfluidics platform

Front Microbiol .. 2023-11; 
Yaping Sun , Danny Hsu Ko , Jie Gao , Kang Fu , Yaping Gao , Qiwen Zhang , Salem Baldi , Tao Hong , Igor Ivanov , Yun He , Hui Tian
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Plasmid DNA Preparation The coding sequences of the D12A, D12A_G197D, SZ_A, SZ_B, SZ_C, and D mutant variants were synthesized and cloned into pet30a using GenScript (Nanjing, China). /Before use, Seq99A synthesized by Genscript (Nanjing, China) was heated in a metal bath at 95°C for 5min and then returned to room temperature for 30min Get A Quote

摘要

In Phi29-α-hemolysin (α-HL) nanopore sequencing systems, a strong electrochemical signal is dependent on a high concentration of salt. However, high salt concentrations adversely affect polymerase activity. Sequencing by synthesis (SBS) requires the use of phi29 polymerase without exonuclease activity to prevent the degradation of modified nucleotide tags; however, the lack of exonuclease activity also affects polymerase processivity. This study aimed to optimize phi29 polymerase for improved salt tolerance and processivity while maintaining its lack of exonuclease activity to meet the requirements of nanopore sequencing. Using salt tolerance compartmentalized self-replication (stCSR) and a microfluidic platf... More

关键词

compartmentalized self-replication (CSR); conservative amino acids; droplet-based microfluidics; salt tolerance polymerase; sequencing by synthesis (SBS).