Construction of Libraries for Methylation Sites by In-gel Competitive Reassociation (IGCR)

The in-gel competitive reassociation (IGCR) procedure was successfully applied to construct a comprehensive library enriched in DNA fragments containing C5mCGG sequences from mouse liver and brain genomic DNA. For IGCR, methylation-insensitive restriction enzyme (Msp I) digests were used as target DNA and methylation-sensitive restriction enzyme (Hpa II) digests as competitor DNA. Southern blot analysis indicated that 60 to 70% of the clones in the library were derived from the methylated sites and overall enrichment was 200- to 1000-fold. IGCR was further applied to construct a library for the sites differentially methylated between brain and liver DNA. In the library, approximately 20% of the Hpa II sites exhibited different degrees of methylation between these tissues.