核酸酶
化学
核酸内切酶
核酸外切酶 III
DNA
核酸外切酶
劈理(地质)
计算生物学
分子生物学
生物化学
生物
基因
DNA聚合酶
大肠杆菌
断裂(地质)
古生物学
作者
Zhihao Ming,Wei Zhang,Ming-Hwai Lin,Xiaofeng Tang,Na Chen,Na Liu,Xiaoyan Xin,Hong‐Bo Wang,Wenpei Xiang,Xianjin Xiao
标识
DOI:10.1021/acs.analchem.1c00507
摘要
Nucleases are powerful tools in various biomedical applications, such as genetic engineering, biosensing, and molecular diagnosis. However, the commonly used nucleases (endonuclease IV, apurinic/apyrimidinic endonuclease-1, and λ exonuclease) are prone to the nonspecific cleavage of single-stranded DNA, making the desired reactions extremely low-yield and unpredictable. Herein, we have developed guiding-strand-controlled nuclease systems and constructed theoretical kinetic models to explain their mechanisms of action. The models displayed excellent agreement with the experimental results, making the kinetics highly predictable and tunable. Our method inhibited the nonspecific cleavage of single-stranded probes while maintaining highly efficient cleavage of double-stranded DNA. We also demonstrated the clinical practicability of the method by detecting a low-frequency mutation in a genomic DNA sample extracted from the blood of a patient with cancer. The limit of detection could be 0.01% for PTEN rs121909219. We believe that our findings provide a powerful tool for the field and the established model provides us a deeper understanding of the enzymatic activities of DNA nucleases.
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