DNA methylation is a mechanism by which the cell regulates gene expression. Methylation is an enzyme-mediated modification that adds a methyl (-CH3) group at a selected site on DNA or RNA. In humans, methylation occurs only at cytosine (C) bases followed by a guanosine (G), known as CpG dinucleotides. The CpG dinucleotides are prone to spontaneous mutations and have been selectively depleted from the mammalian genome. However, some regions of DNA have retained CpG dinucleotides and are referred
to as CpG islands. The CpG islands are found primarily in the 5' region of expressed genes, often in association with promoters. When the promoter CpG island is methylated, the corresponding gene is silenced and transcription does not occur. This is one method of silencing imprinted genes, as the transcription repression is passed on through cell division. Aberrant CpG island methylation of tumor-suppressor genes is frequent in cancer and appears to be an important mechanism of neoplastic transformation.
Methylated DNA can be distinguished from unmethy-lated DNA using sodium bisulfite treatment of DNA, which converts unmethylated C to uracil (U) but leaves methylated C intact.31 This in vitro treatment can then be followed by one of several methods to distinguish C from U, including restriction endonuclease digestion with methy-lation-sensitive enzymes, sequencing, or methylation-specific PCR (MSP).32 MSP of bisulfite-treated DNA uses primer pairs that specifically identify either methylated or unmethylated DNA. The primers are designed to hybridize to regions containing one to three CpG sites concentrated in the 3' region of the primer to increase the specificity of amplification, and enough non-CpG cytosines to ensure that unmodified DNA is not amplified. Gel electrophoresis is used to detect the presence or absence of the amplicon in each of the two reactions, indicating the presence of unmethylated or methylated alleles or both. A novel modification is the use of quantitative MSP, which combines MSP with real-time PCR to distinguish the high-level CpG methylation in neoplasia from low-level methylation that can occur with aging or in nonneoplastic conditions such as metaplasia.33
Examples of Applications of Methylation-Specific PCR
1. Analysis of imprinted genes
2. Clonality assessment based on X chromosome inactivation
3. Abnormal methylation in neoplasia
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