Sequence terminus dependent PCR for site-specific mutation and modification detection.
Gaolian XuHao YangJiani QiuJulien ReboudLinqing ZhenWei RenHong XuJonathan M CooperHongchen GuPublished in: Nature communications (2023)
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).
Keyphrases
- real time pcr
- loop mediated isothermal amplification
- nucleic acid
- genome wide
- dna methylation
- end stage renal disease
- primary care
- wild type
- newly diagnosed
- ejection fraction
- circulating tumor
- small molecule
- gene expression
- transcription factor
- peritoneal dialysis
- prognostic factors
- quality improvement
- patient reported outcomes
- patient reported