This is the classic paper about MSP, a routine technique used in all epigenetics labs. The article includes a detailed and useful description of the careful process of primer design, compared with those for standard PCR and bisulfite sequencing amplification protocols. Curiously, this is also the most cited paper about gene silencing.
Mapping of methylated regions in DNA has relied primarily on Southern hybridization approaches, but [this] requires large amounts of high molecular weight, can detect methylation only if present in greater than a few percent of the alleles and can only provide information about those CpG sites found within sequences recognized by methylation-sensitive restriction enzymes. A more sensitive method of methylation detection combines the use of methylation-sensitive enzymes and PCR. After digestion of DNA with the enzyme, PCR will amplify from primers flanking the restriction site only if DNA cleavage has been prevented by methylation. Like Southern- based approaches, this method can only monitor CpG methylation in methylation-sensitive restriction sites. Moreover, the restriction of unmethylated DNA must be complete, since any uncleaved DNA will be amplified by PCR yielding a false positive result for methylation. This approach has been useful in studying samples where a high percentage of alleles of interest are methylated. However, difficulties in distinguishing between incomplete restriction and low numbers of methylated alleles make this approach unreliable for detection of tumor suppressor gene hypermethylation in small samples or in samples where methylated alleles represent a small fraction of the population. The chemical modification of cytosine to uracil by bisulfite treatment has provided another method for the study of DNA methylation that avoids the use of restriction enzymes. However, this method is technically rather difficult and labor-intensive, and, without cloning of the amplified products, the technique is less sensitive than Southern analysis, requiring -25% of the alleles to be methylated for detection.JG. Herman et al. 1996. Proc Natl Acad Sci USA 93(18):9821-9826
Primers were designed to discriminate between methylated and unmethylated alleles following bisulfite treatment and to discriminate between DNA modified by bisulfite and that which had not been modified. To accomplish this, primer sequences were chosen for regions containing frequent cytosines (to distinguish unmodified from modified DNA), and CpG pairs near the 3' end of the primers (to provide maximal discrimination in the PCR between methylated and unmethylated DNA) [All primers for genomic sequencing were designed to avoid any CpGs in the sequence]. The fragment of DNA to be amplified was intentionally small, to allow the assessment of methylation patterns in a limited region and to facilitate the application of this technique to samples, such as paraffin blocks, where amplification of larger fragments is not possible.PCR with these primers could be performed with or without 5% dimethyl sulfoxide. DNA not treated with bisulfite (unmodified) failed to amplify with either set of methylated or unmethylated specific primers, but readily amplified with primers specific for the sequence before modification.
This technique provides significant advantages over previous PCR-based techniques and other methods used for assaying methylation. MSP is markedly more sensitive than Southern analysis, facilitating detection of low numbers of methylated alleles and the study of DNA from small samples. MSP allows the study of paraffin-embedded materials, which could not previously be analyzed by Southern analysis. MSP also allows examination of all CpG sites, not just those within sequences recognized by methylation-sensitive restriction enzymes. This markedly increases the number of such sites that can be assessed and will allow rapid, fine mapping of methylation patterns throughout CpG-rich regions.MSP also eliminates the frequent false positive results due to partial digestion of methylation-sensitive enzymes inherent in previous PCR methods for detecting methylation. Furthermore, with MSP, simultaneous detection of unmethylated and methylated products in a single sample confirms the integrity of DNA as a template for PCR and allows a semiquantitative assessment of allele types that approximates the quantitation determined by Southern analysis. Finally, the ability to validate the amplified product by differential restriction patterns is an additional advantage. The only technique that can provide more direct analysis than MSP for most CpG sites within a defined region is genomic sequencing. However, MSP can provide similar information and has the following advantages. First, MSP is much simpler and requires less time than genomic sequencing. Second, MSP avoids the use of expensive sequencing reagents: this factor make MSP better suited for the analysis of large numbers of samples. Third, the use of PCR as the step to distinguish methylated from unmethylated DNA in MSP allows for a significant increase in the sensitivity of methylation detection.
