Murgia, Luciano (2014) Approcci analitici per la determinazione del grado di metilazione globale del DNA. Doctoral Thesis.
The term epigenetics was first used to refer to the complex interactions between the genome and the environment that are involved in development and differentiation in higher organisms. Today, this term is used to refer to heritable alterations that are not due to changes in DNA sequence. Rather, epigenetic modifications, or tags, such as DNA methylation and histone modification, alter DNA accessibility and chromatin structure, thereby regulating patterns of gene expression. These processes are crucial to normal development and differentiation of distinct cell lineages in the adult organism. Therefore, the quantification of DNA methylation is an important tool in the study of gene regulation. In particular, tissues stored as paraffin blocks are a potential source of DNA for retrospective genetic and epigenetic analysis. Although tissue architecture and proteins are preserved, extraction of nucleic acids may be difficult, yielding low quantity of degraded DNA. For this reason the evaluation of the methylation degree from formalin-fixed, paraffin-embedded (FFPE) DNA extracts requires methods with high sensitivity and selectivity. We developed the first capillary electrophoresis method able to measure global methylation in FFPE DNA extracts. A field-amplified sample injection capillary electrophoresis method with UV detection for the separation and quantification of cytosine and 5-methylcytosine released following DNA hydrolysis by means of formic acid was employed.
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