Methylation specific PCR (Polymerase Chain Reaction) is used to detect genes or sequences with DNA methylation.
Importance of Detecting DNA Methylation
DNA methylation is an important epigenetic process which has diverse roles to regulate gene expression, imprinted genes, inactivation of X chromosomes, and the dysregulation of these patterns in diseases. Apart from nucleotides, proteins and lipids also get methylated. During DNA replication, a methyl group is added on the pyrimidine ring of cytosine on the 5 position (5-methylcytosine) in methylation (CpG cytosines). This process is mediated by DNA methyltransferases.
Methods to Detect DNA Methylation
Apart from methylation-specific PCR, another method to detect methylation includes southern hybridisation with methylation-sensitive endonucleases. In this method, an endonuclease specific to methylation digests the genomic DNA. Subsequently, it is electrophorized on an agarose gel and a specific DNA probe is used. The endonuclease in this case will cleave the DNA if the cytosines are methylation free. Thus, regions which are methylated remain uncleaved and appear as large bands on the gel. However, this method requires large amounts of DNA for each analysis. Also, only the cytosines for which methylation-specific endonucleases are available can be examined.
Concept of Methylation Specific PCR
In this method, two methylation specific primers are used: an unmethylated primer and a methylated primer. The unmethylated primer amplifies sodium bisulfite converted DNA which is present in unmethylated DNA, whereas the methylated primer amplifies sodium bisulphite converted methylated DNA.
Sodium Bisulphite Treatment
The DNA is subjected to chemical reactions to convert cytosine to uracil and to achieve limited degradation of DNA. For this, the DNA is treated with sodium bisulfite for 16 hours at 50 °C. The amount of DNA should not be increased as it can lead to incomplete conversion and presence of false positives. After treatment with sodium bisulfite, the DNA exists as single strand and is highly sensitive for degradation. This DNA should be stored at -20 °C to avoid degradation and fragmentation.
To discriminate between the methylated and unmethylated DNA optimally, the primers should have three CpG nucleotides in the 3’ segment of the primer. The primers should have a large numbers of non-CpG cytosines in the template. The length of primers should be at least 23-24 bp to get specific annealing of the primer. To achieve good results, the product which has to be amplified should not be larger than 150 bp.
Beta-mercaptoethanol should be used to amplify GC-rich DNA. The annealing temperature of the primers is critical. The correct temperature should be determined empirically by examining several annealing temperatures. Although the typical number of amplification cycles should be around 25 cycles, the specific number of cycles of amplification has to be determined for each MSP reaction. In each experiment, positive, negative, and water controls should be included. After the amplification step, 6−8% nondenatured polyacrylamide gel electrophoresis can be performed to visualise the amplified products.
Applications of MSP
Multiplex Nested MSP
For increasing the sensitivity of MSP to detect methylated DNA, a nested, two-stage MSP was developed. In this method, a first round of PCR is performed to amplify sodium bisulphite modified DNA. The PCR product obtained after the first cycle is further diluted 50−100 fold. Then the sample is subjected to a second round of PCR reactions. Nested MSP is more sensitive and can detect methylation even in samples which have poor DNA quality.
Several quantitative methods have been developed to quantify the level of methylation in the DNA. Quantitative MSP does not include additional PCR steps or gel electrophoresis making this a faster method to screen methylated DNA. This technique is also used to detect aberrations in the methylation of circulating DNA in serum of cancer patients.
In Situ MSP
As the other methods of MSP cannot detect changes during specific stages of development, researchers have combined in situ hybridization with MSP for in situ detection of methylated DNA. In this method, tissue sections are treated with sodium bisulfite and the MSP analysis is done. In situ hybridization is performed on the samples after the amplification step to generate in situ MSP results.