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Sequence terminus dependent PCR for site-specific mutation and modification detection

Author

Listed:
  • Gaolian Xu

    (Shanghai Jiao Tong University)

  • Hao Yang

    (Shanghai Jiao Tong University)

  • Jiani Qiu

    (Shanghai Jiao Tong University)

  • Julien Reboud

    (University of Glasgow)

  • Linqing Zhen

    (Shanghai Jiao Tong University)

  • Wei Ren

    (Shanghai Jiao Tong University)

  • Hong Xu

    (Shanghai Jiao Tong University)

  • Jonathan M. Cooper

    (University of Glasgow)

  • Hongchen Gu

    (Shanghai Jiao Tong University)

Abstract

The detection of changes in nucleic acid sequences at specific sites remains a critical challenge in epigenetics, diagnostics and therapeutics. To date, such assays often require extensive time, expertise and infrastructure for their implementation, limiting their application in clinical settings. Here we demonstrate a generalizable method, named Specific Terminal Mediated Polymerase Chain Reaction (STEM-PCR) for the detection of DNA modifications at specific sites, in a similar way as DNA sequencing techniques, but using simple and widely accessible PCR-based workflows. We apply the technique to both for site-specific methylation and co-methylation analysis, importantly using a bisulfite-free process - so providing an ease of sample processing coupled with a sensitivity 20-fold better than current gold-standard techniques. To demonstrate the clinical applicability through the detection of single base mutations with high sensitivity and no-cross reaction with the wild-type background, we show the bisulfite-free detection of SEPTIN9 and SFRP2 gene methylation in patients (as key biomarkers in the prognosis and diagnosis of tumours).

Suggested Citation

  • Gaolian Xu & Hao Yang & Jiani Qiu & Julien Reboud & Linqing Zhen & Wei Ren & Hong Xu & Jonathan M. Cooper & Hongchen Gu, 2023. "Sequence terminus dependent PCR for site-specific mutation and modification detection," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-36884-4
    DOI: 10.1038/s41467-023-36884-4
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    References listed on IDEAS

    as
    1. Qian Liu & Li Fang & Guoliang Yu & Depeng Wang & Chuan-Le Xiao & Kai Wang, 2019. "Detection of DNA base modifications by deep recurrent neural network on Oxford Nanopore sequencing data," Nature Communications, Nature, vol. 10(1), pages 1-11, December.
    2. Da Jia & Renata Z. Jurkowska & Xing Zhang & Albert Jeltsch & Xiaodong Cheng, 2007. "Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation," Nature, Nature, vol. 449(7159), pages 248-251, September.
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