methylationspecific

Methylation-specific is a term used in molecular biology to describe assays, analyses, or reagents that are specific for the methylation status of nucleic acids, most commonly DNA. In vertebrates, DNA methylation refers mainly to the addition of a methyl group at the 5 position of cytosine in CpG dinucleotides, with regulatory consequences for gene expression and genome stability.

The core principle is to distinguish methylated from unmethylated cytosines by exploiting chemical, enzymatic, or antibody-based differences. Bisulfite treatment converts unmethylated cytosines to uracil, while 5-methylcytosine is relatively resistant; subsequent PCR, sequencing, or array-based readout reveals methylation patterns. Techniques such as methylation-specific PCR (MSP), and its quantitative variant MS-HRM, use primers that differentiate methylated and unmethylated sequences after bisulfite conversion. Bisulfite sequencing or pyrosequencing provides single-base resolution across targeted regions or genome-wide approaches via reduced representation bisulfite sequencing (RRBS) or whole-genome bisulfite sequencing (WGBS).

Alternatively, methylation-specific approaches use enrichment, such as MeDIP, which uses antibodies against 5-methylcytosine to isolate methylated DNA for downstream analysis.

Applications include cancer biomarker discovery, epigenetic profiling, studies of imprinting and development, and environmental epigenetics. The choice of method depends on desired resolution, genome coverage, DNA quality, and quantitative needs.

Limitations include incomplete bisulfite conversion, DNA degradation, PCR bias toward certain sequences, and interpretational complexity when methylation is heterogeneous. Controls and validation across platforms are common.