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Abstract <p>One of the challenges in <i>de novo</i> DNA synthesis is the occurrence of errors. The number of errors associated with synthesis protocols limits the efficiency of obtaining synthetic DNA fragments, both on small and on large scales. In this study, we propose a new rapid protocol for the synthesis of DNA fragments using the thermostable endonuclease V from <i>Thermotoga maritima</i> (Tma endonuclease) as an error correction enzyme. The aim of the research is to develop a one-step error correction method for <i>de novo</i> gene synthesis. The recombinant Tma endonuclease enzyme was obtained. To evaluate the efficiency of error correction using Tma endonuclease, the synthesis of the mTomato gene was performed using various protocols. The efficiency of the enzyme was assessed visually on the basis of the number of fluorescent colonies. To confirm the results, random samples of colonies were selected, and the assembled sequences were sequenced using the Sanger method. It was demonstrated that Tma endonuclease can be used in both the classical three-step and the rapid one-step error correction protocols without loss of activity. It was also established that, with comparable accuracy of DNA fragment synthesis, the efficiency of Tma endonuclease is similar to that of the commercial enzyme Correctase (ThermoFisher). It was concluded that one-step error correction using endonuclease V accelerates the process of DNA fragment synthesis and error correction. By reducing the number of steps, this protocol is better suited for high-throughput DNA synthesis platforms compared to the classical protocol using non-thermostable enzymes.</p>

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Error Correction Occurring during DNA Fragment Synthesis Using Endonuclease V from Thermotoga maritima

  • 期刊论文

  • 发表日期：

    2025年8月29日

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  • D. A. Garagulya,
  • D. V. Evsyutina,
  • I. S. Rog,
  • G. Yu. Fisunov

摘要

Abstract

One of the challenges in de novo DNA synthesis is the occurrence of errors. The number of errors associated with synthesis protocols limits the efficiency of obtaining synthetic DNA fragments, both on small and on large scales. In this study, we propose a new rapid protocol for the synthesis of DNA fragments using the thermostable endonuclease V from Thermotoga maritima (Tma endonuclease) as an error correction enzyme. The aim of the research is to develop a one-step error correction method for de novo gene synthesis. The recombinant Tma endonuclease enzyme was obtained. To evaluate the efficiency of error correction using Tma endonuclease, the synthesis of the mTomato gene was performed using various protocols. The efficiency of the enzyme was assessed visually on the basis of the number of fluorescent colonies. To confirm the results, random samples of colonies were selected, and the assembled sequences were sequenced using the Sanger method. It was demonstrated that Tma endonuclease can be used in both the classical three-step and the rapid one-step error correction protocols without loss of activity. It was also established that, with comparable accuracy of DNA fragment synthesis, the efficiency of Tma endonuclease is similar to that of the commercial enzyme Correctase (ThermoFisher). It was concluded that one-step error correction using endonuclease V accelerates the process of DNA fragment synthesis and error correction. By reducing the number of steps, this protocol is better suited for high-throughput DNA synthesis platforms compared to the classical protocol using non-thermostable enzymes.