Expression of genetic information is related to the presence of 5-methylcytosine in DNA. The distribution of 5-methylcytosine varies in different tissues and its frequency is drastically modified according to the developmental stage of the cell as well as under pathological conditions. Among other applications, the presence of ” 5-methylcytosine distribution is currently used as a marker of carcinogenesis as well as of abnormal neurological and other pathological conditions. Analysis of the distribution and mostly the frequency of this modified base, applying an accurate and reproducible method, is a challenge, frequently met by the application of various methods involving the use of analytical techniques. In this review we present the high-throughput analysis of DNA methylation by High Performance Liquid Chromatography, Denaturing High Performance Liquid Chromatography and Gas Chromatography. Moreover, we discuss in detail the principles, as well as the advantages and disadvantages of the above methods, their application for analyzing either global or gene-specific methylation and the biological issues addressed in these studies. In addition, we discuss the detection, by the same methods, of other minor DNA bases, besides 5- methylcytosine, as well as the choice of base derivatives monitored in these studies. Finally, we present information regarding the evaluation of analytical data relative to those obtained by molecular techniques and discuss the particular clinical applications for which the use of analytical techniques can be particularly valuable.
Keywords: DNA methylation, 5-methylcytosine, HPLC, DHPLC, GC, MS, Drosophila melanogaster, Saccharomyce cerevisiae, Caenorhabdi-tis elegans, Escherichia coli, epigenetic DNA modification, Delta DNMT3B, SNURF-SNRPN, death-associated protein kinase (DAPK, insulin-like growth factor II precursor, IGF2, dihydropyrimidine dehydrogenase, N-methyl-N-nitrosourea, 5-fluorouracil, 2-acetylaminofluorene, COBRA, Combined Bisulfite Restriction Assay, CpG, cytosine and guanine dinucleotide, Denaturing High Performance Liquid Chromatography, ECD, Electron Capture Detector, FID, Flame Ionization Detecto, TBDMS, tert-butyldimethylsilyl, MDS, Myelodysplastic Syndrome, MSP, Metlylation Specific PCR, PCR, Polymerase Chain Reaction