DNA methylation is one of the most important modifications in mammalian cells which plays an essential role in regulating growth of the cells and their proliferation. In humans, it refers to the addition of a methyl group at the 5-carbon position of cytosine (5-methylcytosine or 5 mC), which happens exclusively at symmetric CG sites on the DNA double helix – also termed CpG dinucleotides.
The detection of DNA methylation patterns is a rapidly advancing area of research, with promises the possibility of the methylation profiling to distinguish various tumor and cancer types, and possibly their response to chemotherapeutic agents. Detection of aberrancies of DNA methylation appears to be one of the most significant tests in early cancer diagnosis.
Many techniques developed for detection of DNA methylation can be divided into three groups: chemical modification with bisulfite (represented by bisulfite genomic sequencing), restriction enzyme digestion (represented by methylation-sensitive restriction endonucleases) and affinity-based isolation of methylated DNA (represented by methylated DNA immunoprecipitation).
Bisulfite genomic sequencing
Bisulfite genomic sequencing technology is regarded as a gold-standard in the field for detection of DNA methylation due to its quantitative, qualitative and efficient approach to pinpoint 5-methylcytosine at single base-pair resolution. This technique allows differentiation of methylated from unmethylated DNA via PCR amplification and subsequent analysis of the PCR products.
PCR is a method used to amplify selected sections of DNA for analysis. During PCR amplification in bisulfite genomic sequencing, unmethylated cytosines amplify as thymine, while methylated cytosines amplify as cytosine. The methylation status can be then determined either through direct PCR product sequencing (where average methylation status is detected) or sub-cloning sequencing (when single molecules distribution of methylation patterns is detected).
Most methods for analyzing DNA methylation at the specific loci of interest are based on this approach. Prominent techniques that work on the basis of bisulfite are Combined Bisulfite Restriction Analysis (COBRA), Methylation Specific PCR (MSP) and Methylation-sensitive Single Nucleotide Primer Extension (Ms-SNuPE). Numerous modifications of bisulfite genomic sequencing protocol have been explored to optimize the final result and improve overall accuracy of this procedure.
Methylation-sensitive restriction endonucleases
Methylation-sensitive restriction endonucleases represent classic tools of DNA methylation analysis. Methods that employ them either enrich for methylated DNA or unmethylated DNA. The ability to enrich unmethylated DNA – by digesting methylated DNA or by isolating smaller fragments generated by methylation-inhibited enzymes – is particularly useful for analyzing sizeable, heavily methylated genomes.
The most frequently used restriction enzymes are the restriction enzyme pairs (also called isoschizomers) HpaII and MspI, which recognize the sequence CCGG. HpaII is blocked by methylation of either of the two cytosines, whereas MspI is blocked by methylation of merely the outer cytosine. In mammalian genomes, where methylation is linked almost exclusively to CG sites, HpaII is inhibited and MspI is not.
Over 60% of CG sites are methylated within the human genome, hence enriching unmethylated DNA reduces a complexity of the sample significantly. The procedure itself seems to be simpler and faster than bisulfite genomic sequencing. The important limitation shared by all techniques based on methylation-sensitive restriction endonucleases is the analysis of methylation only within recognition sites.
Methylated DNA immunoprecipitation
Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest. In this procedure genomic DNA is sonicated into fragments and immunoprecipitated with a monoclonal antibodies (antibodies produced by a single clone of cells) that specifically recognize 5-methylcytidine.
Methylated DNA bound to the antibody complex is then separated from the rest of the DNA by a magnet used to pull the complexes out of a solution. After isolation and purification is performed, the methylated DNA is ready for any locus-specific (PCR) or genome-wide (sequencing and microarray) methylation studies. The technique provides a rapid view of DNA methylation levels with only a limited amount of starting DNA.
Although MeDIP is faster than traditional bisulfite sequencing methods and not limited to the analysis of specific sequences like in the restriction enzyme analysis, the immunoprecipitation is dependent on DNA sequence (including CpG density), presence of repetitive elements and composition. Thus, appropriate controls are necessary for the analysis and interpretation of obtained results.