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

Gaolian Xu, Hao Yang, Jiani Qiu, Julien Reboud, Linqing Zhen, Wei Ren, Hong Xu (), Jonathan M. Cooper () and Hongchen Gu ()
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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

Nature Communications, 2023, vol. 14, issue 1, 1-11

Abstract: 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).

Date: 2023
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DOI: 10.1038/s41467-023-36884-4

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