3.1.2. Methods Based on Methylation-Sensitive Restriction Enzymes (MSRE)

MSREs are unable to cleave DNA if their restriction site is methylated. Therefore, methylated DNA stays intact while unmethylated is digested by the RE. They can be used in combination with isoschizomer (enzymes with identical restriction site) insensitive to methylation. The most frequently used pair of enzymes is HpaII (MSRE) and MspI (insensitive isoschizomer) which cleave CCGG restriction site. This approach is often used for CGI analysis. Historically it was the original way of DNA methylation analysis from 1979 [76]. The great advantage of this approach is that it does not require bisulfite conversion, therefore lower DNA input is needed with a considerably easier primer design. The disadvantages, on the other hand, include the fact that only sites with available MSRE can be analyzed, and furthermore if the digestion is incomplete false-positive results are generated. According to the Šestáková et al. the price per sample is quite high [50]. Several detection techniques follow after the restriction enzyme digestion. The older approach involved Southern blot analysis and required input around 10 μg of DNA [77].

A more widely used approach utilizes PCR amplification with primers flanking the restriction site. If the DNA is methylated, digestion does not proceed, leading to successful PCR amplification. Our group successfully applied this approach in determination of E6 gene promoter methylation in human papillomavirus 16 isolated from cervical smears [27]. The modern approach utilizes Real-Time PCR. A great description of this method can be found in [78] where they address enzyme and template amount optimization so that nonspecific restriction and incomplete restriction are both minimized. The disadvantage is that at least 2 CpG RE sites should be present in an amplicon for the assay to be reliable [50]. Overall, this method is suitable for both methylated and unmethylated sites but with a higher frequency of CpG. Moreover, Zymo Research offers OneStep qMethyl-PCR kit which contains reagents and controls for quantitative detection which is ideal for screening several loci if one does not want to optimize their controls.

MS-MPLA is a technique originally based on the HhaI enzyme which cuts GCGC sites with probes also containing this sequence. It is described in greater detail in [79,80]. The advantages of this approach include single cytosine resolution in a multiplex fashion without the need for bisulfite conversion. Moreover, it is semi-quantitative. Downsides of this approach include optimization and design of probes combined with the need for special ligase and capillary gel electrophoresis.