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Identification of DNA variants at ultra-low variant allele frequencies via Taq polymerase cleavage of wild-specific blockers

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Abstract

Detecting mutations related to tumors holds immense clinical significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a challenge for the advancement of low-copy mutant ctDNA genotyping in cancer cases. To address this, a Taqman qPCR strategy to identify rare mutations at low variant allele fractions (VAFs) has been developed. This strategy combines mutant-specific primers with wild-specific blockers. Diverging from other blocker-mediated PCRs, which rely on primer-induced strand displacement or the use of modified oligos resistant to Taq polymerase, our innovation is built upon the cleavage of specific blockers by Taq polymerase. Given its unique design, which does not hinge on strand displacement or base modification, we refer to this novel method as unmodified-blocker cleavage PCR (UBC-PCR). Multiple experiments consistently confirmed that variant distinction was improved significantly by introduction of 5′ unmatched blockers into the reaction. Moreover, UBC-PCR successfully detected mutant DNA at VAFs as low as 0.01% across six different variant contexts. Multiplex UBC-PCR was also performed to identify a reference target and three mutations with a sensitivity of 0.01% VAFs in one single tube. In profiling the gene status from 12 lung cancer ctDNA samples and 22 thyroid cancer FNA DNA samples, UBC-PCR exhibited a 100% concordance rate with ddPCR and a commercial ARMS kit, respectively. Our work demonstrates that UBC-PCR can identify low-abundance variants with high sensitivity in multiplex reactions, independent of strand displacement and base modification. This strategy holds the potential to significantly impact clinical practice and precision medicine.

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Acknowledgements

This work was supported by grants from Natural Science Foundation of China (NSFC) (82172954, 81872056, 82003581, and 81903391), Natural Science Foundation of Jiangsu Province (BK20190148), “333” Engineering Project Jiangsu province ((2022) 2-060), Jiangsu Young Medical Talents (QNRC2016188), Taihu Talent Plan (JZ), Reserve Talents of Double Hundred Talent Plan (HB2020017, HB2020018), General Program of Jiangsu Commission of Health (M2020012), Wuxi Medical Innovation Team (CXTD2021006), Wuxi Science and Technology Development Fund (N20192048), and Cohort and Clinical Research Program of Wuxi Medical Center, Nanjing Medical University (WMCC202304). Jiangsu Association for Science and Technology Support Project for Young Scientific and Technological Talents also supported this work. We thank Clarity Manuscript Consultants for assistance with language editing.

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Authors and Affiliations

  1. Department of Laboratory Medicine, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi People’s Hospital, Wuxi Medical Center, Nanjing Medical University, 299 Qingyang Road, Wuxi, 214023, Jiangsu, China

    Zhaocheng Liu, Rui Zhang, Li Ji, Lingli Gong, Ying Yin, Koukou Li, Zhening Pu & Jian Zou

  2. Center of Clinical Research, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi People’s Hospital, Wuxi Medical Center, Nanjing Medical University, Wuxi, 214023, Jiangsu, China

    Zhaocheng Liu, Rui Zhang, Li Ji, Lingli Gong, Ying Yin, Koukou Li, Zhening Pu & Jian Zou

  3. Department of Neurosurgery, The Affiliated Wuxi Second Hospital of Nanjing Medical University, 68 Zhongshan Road, Wuxi, 214122, Jiangsu Province, China

    Xiushuai Li & Qing Wang

  4. Department of Thoracic Surgery, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi People’s Hospital, Wuxi Medical Center, Nanjing Medical University, Wuxi, 214023, Jiangsu, China

    Yong Ji

  5. Department of Ultrasound, The Affiliated Wuxi People’s Hospital of Nanjing Medical University, Wuxi People’s Hospital, Wuxi Medical Center, Nanjing Medical University, Wuxi, 214023, Jiangsu, China

    Fengsheng Zhou

  6. Department of Pathology, Jiangnan University Medical Center, Wuxi, 214023, Jiangsu, China

    Ping Sun

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  1. Zhaocheng Liu
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Liu, Z., Li, X., Zhang, R. et al. Identification of DNA variants at ultra-low variant allele frequencies via Taq polymerase cleavage of wild-specific blockers. Anal Bioanal Chem 415, 6537–6549 (2023). https://doi.org/10.1007/s00216-023-04931-0

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