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Research articles

ScienceAsia 50 (2024):ID 2024015 1-6 |doi: 10.2306/scienceasia1513-1874.2024.015


Chemical modification and blocking ratio assessment of Taq DNA polymerases using a 2,4,6-trinitrobenzenesulfonic acid (TNBS)-based assay


Jin Wanga, Wenhao Hua, Heqing Wanga, Hong Daia, Guochun Wanga, Zhidan Luoa,b,*, Henghao Xua,b,*

 
ABSTRACT:     Mainstreaming modification approaches of commercial hot-start Taq DNA polymerases include antibody modification, nucleic acid adaptor modification, and chemical modification, among which the chemical modification possesses merits of lower cost and higher efficiency excluding animal-derived matters compared with the former two. In chemical modification, the overall modification ratio of lysine residues is a key parameter to indicate the blocking ratio of modified enzyme without compromising its stability. In this study, 2,4,6-trinitrobenzenesulfonic acid (TNBS)-based assay was applied for chemically modified Taq DNA polymerase activity blocking ratio for the first time. The results showed that at the modification ratio of about 50%, the enzyme activity was completely blocked. This detection method?s features were easy-operation and time-saving for the screening of optimized anhydride modified Taq DNA polymerase in multiple incubation conditions. Notably, this new protocol provided a practical reference for identification of novel chemical compounds that can modify DNA polymerases, thereby facilitating the development of advanced enzymes.

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a Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang 222005 China
b Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang 222005 China

* Corresponding author, E-mail: lzd@jou.edu.cn, xuhh@jou.edu.cn

Received 10 Jan 2023, Accepted 20 May 2023