DNA Methylation Analysis via Restriction Enzyme Digestion and Ligation

The invention extracts only the necessary information for methylation analysis by using restriction enzyme digestion to target specific recognition sites. Instead of processing entire genomes with bisulfite treatment, the method isolates and analyzes only the relevant DNA fragments containing methylation sites, thereby avoiding the loss of mappability information while maintaining methylation detection capability

The invention applies local quality by focusing analysis on specific local regions (restriction enzyme recognition sites) rather than treating the entire genome uniformly. By using methylation-sensitive restriction enzymes that specifically recognize and cut at methylated cytosine sites, the method achieves high-resolution methylation detection at targeted locations without the need for genome-wide bisulfite conversion that causes information loss

The invention applies universality by using a single restriction enzyme that can recognize and cut multiple different DNA sequences containing methylated cytosines. The enzyme HpaII, for example, recognizes the sequence CCGG and can detect methylation at any cytosine position within this pattern throughout the genome, making the method universally applicable to all CpG sites that match the recognition pattern, not limited to specific predetermined regions

The invention changes the analytical parameter from requiring specific enzyme pairs with identical recognition sequences to using a single enzyme's cutting pattern. By analyzing the presence or absence of restriction sites and the sizes of resulting fragments, the method quantifies methylation levels across diverse genomic regions, transforming the limitation of single-enzyme specificity into an advantage for broad genomic coverage

The invention segments the genome into manageable restriction enzyme fragments and analyzes methylation patterns by examining the sizes and compositions of these fragments. By digesting DNA with restriction enzymes and separating the resulting fragments through gel electrophoresis or size-based sequencing, the method efficiently analyzes methylation across the entire genome using relatively simple and cost-effective techniques compared to genome-wide bisulfite sequencing

The invention uses PCR amplification to copy and amplify specific DNA fragments containing restriction sites before analysis. This allows multiple copies of the same genomic region to be generated from limited starting material, enabling repeated analysis and quantification without requiring large amounts of original DNA, thereby reducing overall assay costs while maintaining analytical precision