Methylation Specific PCR
Methylation of cytosine in the 5’ position of the pyrimidine ring is a major modification of the DNA in most organisms. The distribution and number of CH3-Cytosines along the DNA is heritable, but can also change with the developmental state of the cell and as a response to modifications of the environment. The gene silencing effect methylation can have in eukaryotic cells and the discovery of changes in the methylation level during cancer development has increased the interest in this field.

In normal cells methylation occurs predominantly in CG-poor regions, while CG-rich areas, called CpG-islands remain unmethylated. The exception is extensive methylation of CpG islands associated with transcriptional inactivation of regulatory
regions of imprinted genes (such as those associated with Prader-Willi/Angelman syndrome) and genes on the inactive X-chromosome of females. Aberrant methylation of normally unmethylated CpG islands has been documented as a relatively
frequent event in immortalized and transformed cells
and has been associated with transcriptional inactivation of defined tumour suppressor genes in human cancers. Examples of genes that exhibit the characteristics of hypermethylation include: p16, p15, Von Hippel Lindau (VHL) and E-cadherin.

Methylation Specific PCR (MSP) is a new technique enabling the precise mapping of DNA methylation patterns in CpG islands utilizing small amounts of DNA. It offers a universal
and highly sensitive approach. MSP detection has the potential to define tumor suppressor gene function and provides a new strategy for early tumor detection research. In addition, it conveys the methylation status of imprinted genes which supplements cytogenetic analysis of inherited diseases. MSP is a breakthrough in speed and sensitivity for gene methylation
analysis. The procedure takes advantages of the bisulfite-mediated chemical conversion of cytosine to uracil, followed by PCR using primers designed to distinguish methylated from
unmethylated DNA.

General diagram of methylation specific PCR: Cytosines 5’ to guanine are either methylated or unmethylated in the example. Typically, cytosines 5’ to A, C or T are not methylated. The first step in MSP (A) involves the chemical conversion of all unmethylated cytosines to uracil, using soldium bisulfite (CpGenome™ DNA Modification Kit). Methylated cytosines remain unaltered in the process. After chemical conversion, PCR is performed using primers designed to distinguish methylated from unmethylated DNA. Primer design is a critical component of the procedure in that the amplification must be specific. (B) Primers must be designed so that mismatches are created which prevent mispriming between the primer sets and undesired targets. If the sample DNA was originally unmethylated, a product will be produced after PCR using only the U primer set. Conversely, only DNA that is originally methylated can be amplified with the M primer set.