Methylation-specific PCR.

Methylation-specific polymerase chain reaction (MSP) is a technique that has facilitated the detection of promoter hypermethylation at CpG islands in cell lines and clinical samples, including fresh/frozen tissues. The ability of MSP to differentiate methylated from unmethylated cytosine is dependent upon sodium bisulfite treatment of DNA which retains the methylation marks of cytosines together with the specific amplification of this modified DNA using primer sets complimentary only to the formerly methylated or unmethylated alleles. Nested-MSP (MN-MSP) is an alternative method that overcomes the limitations of MSP, especially when it comes to analyzing samples with low quality/quantity of starting DNA (e.g., paraffin-embedded specimens). MN-MSP includes a first round of amplification using primers unbiased toward the methylation status of a single (MN-MSP) or multiple (multiplex MN-MSP) genes followed by conventional MSP. Although MSP and NM-MSP are simple techniques that can easily be incorporated in most molecular biology laboratories, the ability to accurately determine the promoter methylation status of genes largely depends upon the careful design of MSP primers as well as other steps outlined in this chapter.