Merck Millipore CpGenome Universal Unmethylated DNA Set

Methylation of cytosines located 5' to guanosine is known to have a profound effect on the expression of many eukaryotic genes. In normal cells methylation occurs predominantly in CG-poor regions, while CG-rich areas, called CpG-islands remain unmethylated. The exceptions are the extensive methylation of CpG islands associated with transcriptional inactivation of regulatory regions of imprinted genes 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 tumor suppresser genes in human cancers. Hundreds of CpG islands are now known to exhibit the characteristic of hypermethylation in tumors.
Several methods have been developed to determine the methylation status of cytosine. These include digestion with methylation sensitive restriction enzymes as in restriction landmark genomic scanning, oligonucleotide arrays, genomic DNA sequencing and methylation specific PCR (MSP). Some techniques are more useful for discovery while others are better used for monitoring of known methylated cytosines. Genomic DNA sequencing, although time consuming and labor intensive, offers a more universal detection method. MSP is now an established technology for the monitoring of abnormal gene methylation in selected gene sequences. Utilizing small amounts of DNA, this procedure offers sensitive and specific detection of 5-methylcytosine in promoters. It is being exploited to define tumor suppresser gene function, and to provide a new strategy for early tumor detection.
The initial step of both genomic sequencing and MSP is to perform a bisulfite modification of the DNA sample. MSP then involves PCR amplification with specific primers designed to distinguish methylated from unmethylated DNA.