Identifying 5-methylcytosine and related modifications in DNA genomes

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Abstract
Intense interest in the biological roles of DNA methylation, particularly in eukaryotes, has produced at least eight different methods for identifying 5-methylcytosine and related modifications in DNA genomes. However, the utility of each method depends not only on its simplicity but on its specificity, resolution, sensitivity and potential artifacts. Since these parameters affect the interpretation of data, they should be considered in any application. Therefore, we have outlined the principles and applications of each method, quantitatively evaluated their specificity, resolution and sensitivity, identified potential artifacts and suggested solutions, and discussed a paradox in the distribution of m5C in mammalian genomes that illustrates how methodological limitations can affect interpretation of data. Hopefully, the information and analysis provided here will guide new investigators entering this exciting field.