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
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.