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

The detection of changes in nucleic acid sequences at specific sites remains a critical challenge in epigenetics, diagnostics and therapeutics. In this project, we demonstrated 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 applied 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. We also demonstrated the clinical applicability through the detection of single base mutations with high sensitivity and no-cross reaction with the wild-type background.