Methylation Specific PCR

Protocol written by James Herman*

Methylation Specific PCR (MSP) is a simple rapid and inexpensive method to determine the methylation status of CpG islands. This approach allows the determination of methylation patterns from very small samples of DNA, including those obtained from paraffin-embedded samples, and can be used in the study of abnormally methylated CpG islands in neoplasia, in studies of imprinted genes, and in studies of human tumors for clonality by studying genes inactivated on the X chromosome.

MSP utilizes the sequence differences between methylated alleles and unmethylated alleles which occur after sodium bisulfite treatment. The frequency of CpG sites in CpG facilitate this sequence difference. Primers for a given locus are designed which distinguish methylated from unmethylated DNA in bisulfite-modified DNA. Since the distinction is part of the PCR amplification, extraordinary sensitivity, typically to the detection of 0.1% of alleles can be achieved, while maintaining specificity. Results are obtained immediately following PCR amplification and gel electrophoresis, without the need for further restriction or sequencing analysis. MSP also allows the analysis of very small samples, including paraffin-embedded and microdissected samples.

BASIC PROTOCOL

DNA is modified by sodium bisulfite treatment converting unmethylated, but not methylated, cytosines to uracil. Following removal of bisulfite and completion of the chemical conversion, this modified DNA is used as a template for PCR. Two PCR reactions are performed for each DNA sample, one specific for DNA originally methylated for the gene of interest, and one specific for DNA originally unmethylated. PCR products are separated on 6-8% non-denaturing polyacrylamide gels and the bands are visualized by staining with ethidium bromide. The presence of a band of the appropriate molecular weight indicates the presence of unmethylated, and/or methylated alleles, in the original sample.

•  Prepare the PCR mixes

Thaw 10x PCR buffer, NTP’s and primers. Determine the number of samples to be analyzed, including a positive control for both the unmethylated and methylated reactions, and a water control. Make a master mix for each PCR reaction (methylated and unmethylated). For each 50