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

Xu, G., Yang, H., Qiu, J., Reboud, J. , Zhen, L., Ren, W., Xu, H., Cooper, J. M. and Gu, H. (2023) Sequence terminus dependent PCR for site-specific mutation and modification detection. Nature Communications, 14, 1169. (doi: 10.1038/s41467-023-36884-4) (PMID:36859350) (PMCID:PMC9978023)

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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).

Item Type:Articles
Additional Information:This work was supported by the SJTU STAR Interdisciplinary Important Program (20190201, to H.G.), Innovation Research Plan from the Shanghai Municipal Education Commission (ZXWF082101/056, to G.X.), Natural Science Foundation of Shanghai Exploratory Project (19ZR1476000 to G.X.), key projects of special development funds for Shanghai Zhangjiang National Innovation Demonstration Zone (201905-XH-CHJ-H25-201 to H.G.), as well as the UK Research and Innovation through the Medical Research Council (MR/V035401/1 to J.M.C.) and the Biotechnology and Biological Sciences Research Council (BB/T012528/1 to J.M.C.).
Glasgow Author(s) Enlighten ID:Cooper, Professor Jonathan and Reboud, Dr Julien and Xu, Mr Gaolian
Authors: Xu, G., Yang, H., Qiu, J., Reboud, J., Zhen, L., Ren, W., Xu, H., Cooper, J. M., and Gu, H.
College/School:College of Science and Engineering > School of Engineering > Biomedical Engineering
Journal Name:Nature Communications
Publisher:Nature Research
ISSN (Online):2041-1723
Copyright Holders:Copyright © 2023 The Authors
First Published:First published in Nature Communications 14: 1169
Publisher Policy:Reproduced under a Creative Commons License
Data DOI:10.5525/gla.researchdata.1298

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
311995COVIDJonathan CooperMedical Research Council (MRC)MR/V035401/1ENG - Biomedical Engineering
309023Field-based, point-of-need pathogen detection in shrimp farms towards sustainability and food securityJulien ReboudBiotechnology and Biological Sciences Research Council (BBSRC)BB/T012528/1ENG - Biomedical Engineering