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Specificity Enhancement of Deoxyribonucleic Acid Polymerization for Sensitive Nucleic Acid Detection.
Hu, Bei; Wang, Yitao; Sun, Shichao; Luo, Guangcheng; Zhang, Shun; Zhang, Jun; Chen, Lu; Huang, Zhen.
  • Hu B; Key Laboratory of Bio-Resource and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, P. R. China.
  • Wang Y; Key Laboratory of Bio-Resource and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, P. R. China.
  • Sun S; Key Laboratory of Bio-Resource and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, P. R. China.
  • Luo G; Key Laboratory of Bio-Resource and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, P. R. China.
  • Zhang S; Key Laboratory of Bio-Resource and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, P. R. China.
  • Zhang J; Key Laboratory of Bio-Resource and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, P. R. China.
  • Chen L; Szostak-CDHT Institute for Large Nucleic Acids, Chengdu 610041, Sichuan, P.R. China.
  • Huang Z; Key Laboratory of Bio-Resource and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu 610064, Sichuan, P. R. China.
Anal Chem ; 92(24): 15872-15879, 2020 12 15.
Article in English | MEDLINE | ID: covidwho-943827
ABSTRACT
Specificity of DNA polymerization plays a critical role in DNA replication and storage of genetic information. Likewise, biotechnological applications, such as nucleic acid detection, DNA amplification, and gene cloning, require high specificity in DNA synthesis catalyzed by DNA polymerases. However, errors in DNA polymerization (such as mis-incorporation and mis-priming) can significantly jeopardize the specificity. Herein, we report our discovery that the specificity of DNA enzymatic synthesis can be substantially enhanced (up to 100-fold higher) by attenuating DNA polymerase kinetics via the phosphorothioate dNTPs. This specificity enhancement allows convenient and sensitive nucleic acid detection, polymerization, PCR, and gene cloning with complex systems (such as human cDNA and genomic DNA). Further, we found that the specificity enhancement offered higher sensitivity (up to 50-fold better) for detecting nucleic acids, such as COVID-19 viral RNAs. Our findings have revealed a simple and convenient strategy for facilitating specificity and sensitivity of nucleic acid detection, amplification, and gene cloning.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: DNA / RNA, Viral Type of study: Diagnostic study Limits: Humans Language: English Journal: Anal Chem Year: 2020 Document Type: Article

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Full text: Available Collection: International databases Database: MEDLINE Main subject: DNA / RNA, Viral Type of study: Diagnostic study Limits: Humans Language: English Journal: Anal Chem Year: 2020 Document Type: Article