ABSTRACT Methylation of cytosines in the 5’ position of the pyrimidinic ring is the most important epigenetic alteration in eukaryotes. In animals, methylcytosine is mainly found in cytosine-guanine (CpG) dinucleotides, whereas in plants it is more frequently located in cytosine-any base-guanine (CpNpGp) trinucleotide sequences. The presence of 5-methylcytosine (mC) in the promoter of specific genes alters the binding of transcriptional factors and other proteins to DNA in plants and animals. Thus, methylation of C residues in genomic DNA plays a key role in the regulation of gene expression. DNA methylation research can be approached from several standpoints since there are a wide range of techniques available for the study of the occurrence and localization of methylcytosine in the genome. Each technique has its own peculiarities implying that there is a most suitable technique for each specific problem. In any case, DNA methylation status can only be established by the joint use of various methodologies. The available methods for studying the degree of DNA methylation can be classified with respect to the type of information produced: degree of whole genomic DNA methylation, DNA methylation status of a specific sequence, and how to find new methylation hot-spots. Level of methylcytosine occurrence in the genomic DNA can be measured by high-performance separation techniques or by enzymatic/chemical means. The latter are never as sensitive as the former and sometimes their resolution is restricted to endonuclease cleavage sites. Despite the drawbacks, enzymatic/chemical approaches are still commonly used since, unlike separation techniques, they do not require expensive and complex equipment, which is not always available. When separation devices are available, high-performance capillary electrophoresis (HPCE) may be the best choice since it is faster, cheaper and more sensitive than HPLC. Finally, two main alternatives are currently being used to study the degree of DNA methylation in particular DNA sequences: non-bisulfite and bisulfite methods. Global methylcytosine content in DNA both during ageing-reinvigoration in plants and in human tumors are properly presented.
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