Methylation-Specific PCR Protocol and Method
MSP can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of cloning or methylation-sensitive restriction enzymes. The assay consists of initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA.
MSP requires very small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed in DNA extracted paraffin-embedded samples (Herman et al., 1996).
METHODS and MATERIALS: A Protocol for Methylation-Specific PCR, MSP
One micro g of sodium bisulfite-treated genomic DNA is used for PCR amplification using MSP primers (Li et al., 2001) (MSP-Methylated and MSP-Unmethylated). The methylation-specific primers included in which nucleotides corresponding to potentially methylated cytosines where retained.
The primer combination to amplify unmethylated DNA included in which the nucleotides corresponding to cytosine nucleotides were changed to thymine (sense primer) or adenine (antisense primer) (Herman et al., 1996). The PCR conditions are as follows: 1 cycle of 95oC for 15 minutes; 40 cycles of 95 o C for 30 seconds, 50 o C for 30 seconds, and 72 o C for 45 seconds; and 1 cycle of 72 o C for 5 minutes.
The methylation-specific and nonmethylated DNA-specific primers yield different PCR products, respectively, when constructed. Depending on the primers created, differences in PCR temperatures and cycle optimizations are necessary.