Protocol for detecting cancer in blood using targeted DNA methylation

Here, we present a protocol for detecting and differentiating colorectal and gastric cancers using plasma cell-free DNA (cfDNA) with targeted methylated CpG tandem amplification and sequencing (tMCTA-seq), a targeted DNA methylation sequencing approach. We describe steps for sample collection, DNA extraction, bisulfite conversion, and targeting 110 methylated sites by coupling locus-specific primers with a universal CGCGCGG primer. We then detail an optional workflow that adapts tMCTA-seq to other cancer types. This approach achieves high sensitivity, below one haploid-genome equivalent, enabling non-invasive cancer detection.