DNA methylation: a profile of methods and applications.

Ever since methylcytosine was found in genomic DNA, this epigenetic alteration has become a center of scientific attraction, especially because of its relation to gene silencing in disease. There is currently a wide range of methods designed to yield quantitative and qualitative information on genomic DNA methylation. The earliest approaches were concentrated on the study of overall levels of methylcytosine, but more recent efforts havefocused on the study ofthe methylation status of specific DNA sequences. Particularly, optimization of the methods based on bisulfite modification of DNA permits the analysis of limited CpGs in restriction enzyme sites (e.g., combined bisulfite restriction analyses and methylation-sensitive single nucleotide primer extension) and the overall characterization based on differential methylation states (e.g., methylation-specific PCR, MethyLight, and methylation-sensitive single-stranded conformational polymorphism) and allows very specific patterns of methylation to be revealed (bisulfite DNA sequencing). In addition, novel methods designed to search for new methylcytosine hot spots have yielded further data without requiring prior knowledge of the DNA sequence. We hope this review will be a valuable tool in selecting the best techniques to address particular questions concerning the cytosine methylation status of genomic DNA.