Southern analysis using methyl-sensitive restriction enzymes

Methylation of the cytosine base in cytosine-guanine (CG) dinucleotides of genomic DNA is likely to be one of the primary epigenetic "imprints" that results in parental allele-specific expression of certain mammalian genes (1). Despite the increasing popularity of bisulphite sequencing (2), ligationmediated polymerase chain reaction (PCR) (3), the availability of antibodies to 5-methylcytosine ((5m)CG) (4), and more recent developments in DNA array technology (5), Southern analysis using methyl-sensitive DNA restriction enzymes remains one of the more popular methods for the detection and analysis of mammalian DNA methylation. However, it has two significant disadvantages. First, relatively large amounts of genomic DNA are required for Southern blotting, thereby reducing its usefulness in the study of DNA methylation in the female germline and preimplantation embryos. Second, the analysis is limited to CGs that occur in a DNA sequence context that is recognized by a methyl-sensitive restriction enzyme. However, despite these drawbacks, the fact that the technique is still widely used is testament to its robustness, reproducibility, and relative simplicity.