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<p>Considering the widespread use of blood samples in clinical medicine, disease diagnosis, and forensic science, there is an urgent need for the development of Taq DNA polymerases that are resistant to whole blood inhibitors and can maintain high amplification efficiency. This study reveals a dual mechanism by which blood inhibits PCR amplification: first, heme inhibits its activity by binding to DNA polymerase; second, immunoglobulin G binding to template DNA leads to a significant increase in the threshold cycle (<i>C</i>t) value. Meanwhile, high concentrations of inhibitors generally led to a fluorescence quenching effect of the dye during amplification. Based on protein engineering techniques, this study identified key residues in Taq DNA polymerase that interact with heme. The mutant Taq43(TaqR37A/K314A/P387L), constructed by semi-rational design, had a heme-tolerant concentration (7 µmol/L) that was sevenfold higher than that of the wild type (WT). Further, by deleting the highly flexible 5′-3′ exonuclease structural domain and fusing it with the double-stranded DNA-binding protein Sso7d to form a chimera, the final S-KLTaq43 mutant was obtained to achieve robust PCR amplification in the presence of 50% (v/v) whole blood. This study holds significant practical value for advancing the development of direct testing technologies for blood samples.</p>

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Protein engineering-based modification of Taq DNA polymerase resistant to whole blood inhibitors

  • 期刊论文

  • 发表日期：

    2026年4月13日

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  • Zigang Zhang,
  • Hengwei Zhang,
  • Cheng Lu,
  • Siyuan Tian,
  • Zhiming Rao,
  • Xian Zhang

摘要

Considering the widespread use of blood samples in clinical medicine, disease diagnosis, and forensic science, there is an urgent need for the development of Taq DNA polymerases that are resistant to whole blood inhibitors and can maintain high amplification efficiency. This study reveals a dual mechanism by which blood inhibits PCR amplification: first, heme inhibits its activity by binding to DNA polymerase; second, immunoglobulin G binding to template DNA leads to a significant increase in the threshold cycle (Ct) value. Meanwhile, high concentrations of inhibitors generally led to a fluorescence quenching effect of the dye during amplification. Based on protein engineering techniques, this study identified key residues in Taq DNA polymerase that interact with heme. The mutant Taq43(TaqR37A/K314A/P387L), constructed by semi-rational design, had a heme-tolerant concentration (7 µmol/L) that was sevenfold higher than that of the wild type (WT). Further, by deleting the highly flexible 5′-3′ exonuclease structural domain and fusing it with the double-stranded DNA-binding protein Sso7d to form a chimera, the final S-KLTaq43 mutant was obtained to achieve robust PCR amplification in the presence of 50% (v/v) whole blood. This study holds significant practical value for advancing the development of direct testing technologies for blood samples.