DNA methylation is a modification of DNA that occurs after synthesis of the strand. A methyl group (CH3) is added to one of the four nucleotides in DNA, specifically cytosine, by the enzyme DNA methyltransferase. This chemical change has been found to occur in CpG islands, areas of the genome rich in cytosines and guanines, cytosine’s complement. Individual genomes have different DNA methylation patterns, which is referred to as the epigenome.
Epigenetic patterns are associated with the activation and/or inactivation of genes and the X chromosome, which results in oncogene, tumor suppressor and imprinting processes. Because of the potential disease associations (e.g., cancer), researchers have been very interested in studying the methylation process and patterns, particularly changes in the amount of methylation and the genes in which the changes occur.
Methods used to study DNA methylation exploit the chemical properties of the nucleotides and the fact that methyl groups are not usually found on the DNA strands outside of this epigenetic modification. A major technique in studying specific genes or DNA regions is polymerase chain reaction (PCR), which uses the cell’s own replication mechanism to amplify portions of DNA. Various techniques are coupled with this basic methodology to determine the pattern of methylation.
One method uses restriction enzymes that recognize the methylated sequence, digesting the DNA at points where a methyl group is present. A second set of methods use sodium bisulfite treatment to convert unmethylated cytosines to uracil via deamination. PCR amplification results in A:T pairs where G:C pairs were present in the original DNA, allowing detection by sequencing of the PCR product, Southern blot, or fluorescent probes with trademarked Taqman technology. In yet another method, methylation-specific PCR uses primers specific for methylated cytosine.
Global methylation analysis measures the overall amount of methylated cytosines in the genome. Some new genome-wide scanning techniques use sodium bisulfite treatment. Yet others use bisulfite-independent microarray analysis. Methyl-cytosine specific antibodies can be used in binding assays to determine the overall amount of methylation, which is helpful for qualitative studies of hypermethylation and hypomethylation in diseased states versus normal.
Chromatographic methods are also used, such as high performance liquid chromatography (HPLC). The technique separates the nucleotides by size, allowing quantification of the relative amount of methylated cytosine in the DNA sample. Liquid chromatography coupled with mass spectrometry is another, similar technique, but is best used for gene-specific PCR amplified regions of DNA. Yet another technique is methyl accepting capacity assay, which uses radiographic chromatography to determine the relative binding of methyl groups to the DNA as an indicator of the relative non-methylation of the sample. A review in 2002 outlined the numerous chromatographic methods used by researchers to study DNA methylation.
Over the last two decades, a number of reagent kits have become commercially available, making studies more efficient and standardized, and the field of epigenetics has grown in the last decade, leading to more specific and effective methods of studying DNA methylation. As researchers understand more about the role methyl-cytosine plays in gene regulation, they develop new techniques to measure the specific properties associated with the process of interest.
