<P> The restriction enzyme DpnI can recognize 5' - GmeATC - 3' sites and digest the methylated DNA . Being such a short motif, it occurs frequently in sequences by chance, and as such its primary use for researchers is to degrade template DNA following PCRs (PCR products lack methylation, as no methylases are present in the reaction). Similarly, some commercially available restriction enzymes are sensitive to methylation at their cognate restriction sites, and must as mentioned previously be used on DNA passed through a dam - / dcm - strain to allow cutting . </P> <P> DNA methylation can be detected by the following assays currently used in scientific research: </P> <Ul> <Li> Mass spectrometry is a very sensitive and reliable analytical method to detect DNA methylation . MS in general is however not informative about the sequence context of the methylation, thus limited in studying the function of this DNA modification . </Li> <Li> Methylation - Specific PCR (MSP), which is based on a chemical reaction of sodium bisulfite with DNA that converts unmethylated cytosines of CpG dinucleotides to uracil or UpG, followed by traditional PCR . However, methylated cytosines will not be converted in this process, and primers are designed to overlap the CpG site of interest, which allows one to determine methylation status as methylated or unmethylated . </Li> <Li> Whole genome bisulfite sequencing, also known as BS - Seq, which is a high - throughput genome - wide analysis of DNA methylation . It is based on aforementioned sodium bisulfite conversion of genomic DNA, which is then sequenced on a Next - generation sequencing platform . The sequences obtained are then re-aligned to the reference genome to determine methylation states of CpG dinucleotides based on mismatches resulting from the conversion of unmethylated cytosines into uracil . </Li> <Li> Reduced representation bisulfite sequencing, also known as RRBS knows several working protocols . The first RRBS protocol was called RRBS and aims for around 10% of the methylome, a reference genome is needed . Later came more protocols that were able to sequence a smaller portion of the genome and higher sample multiplexing . EpiGBS was the first protocol were you could multiplex 96 sample in one lane of Illumina sequencing and were a reference genome was not longer needed . A de novo reference construction from the Watson and Crick reads made population screening of SNP's and SMP's simultaneously a fact . </Li> <Li> The HELP assay, which is based on restriction enzymes' differential ability to recognize and cleave methylated and unmethylated CpG DNA sites . </Li> <Li> GLAD - PCR assay, which is based on new type of enzymes--site - specific methyl - directed DNA endonucleases, which hydrolyze only methylated DNA . </Li> <Li> ChIP - on - chip assays, which is based on the ability of commercially prepared antibodies to bind to DNA methylation - associated proteins like MeCP2 . </Li> <Li> Restriction landmark genomic scanning, a complicated and now rarely used assay based upon restriction enzymes' differential recognition of methylated and unmethylated CpG sites; the assay is similar in concept to the HELP assay . </Li> <Li> Methylated DNA immunoprecipitation (MeDIP), analogous to chromatin immunoprecipitation, immunoprecipitation is used to isolate methylated DNA fragments for input into DNA detection methods such as DNA microarrays (MeDIP - chip) or DNA sequencing (MeDIP - seq). </Li> <Li> Pyrosequencing of bisulfite treated DNA . This is sequencing of an amplicon made by a normal forward primer but a biotinylated reverse primer to PCR the gene of choice . The Pyrosequencer then analyses the sample by denaturing the DNA and adding one nucleotide at a time to the mix according to a sequence given by the user . If there is a mis - match, it is recorded and the percentage of DNA for which the mis - match is present is noted . This gives the user a percentage methylation per CpG island . </Li> <Li> Molecular break light assay for DNA adenine methyltransferase activity--an assay that relies on the specificity of the restriction enzyme DpnI for fully methylated (adenine methylation) GATC sites in an oligonucleotide labeled with a fluorophore and quencher . The adenine methyltransferase methylates the oligonucleotide making it a substrate for DpnI . Cutting of the oligonucleotide by DpnI gives rise to a fluorescence increase . </Li> <Li> Methyl Sensitive Southern Blotting is similar to the HELP assay, although uses Southern blotting techniques to probe gene - specific differences in methylation using restriction digests . This technique is used to evaluate local methylation near the binding site for the probe . </Li> <Li> MethylCpG Binding Proteins (MBPs) and fusion proteins containing just the Methyl Binding Domain (MBD) are used to separate native DNA into methylated and unmethylated fractions . The percentage methylation of individual CpG islands can be determined by quantifying the amount of the target in each fraction . Extremely sensitive detection can be achieved in FFPE tissues with abscription - based detection . </Li> <Li> High Resolution Melt Analysis (HRM or HRMA), is a post-PCR analytical technique . The target DNA is treated with sodium bisulfite, which chemically converts unmethylated cytosines into uracils, while methylated cytosines are preserved . PCR amplification is then carried out with primers designed to amplify both methylated and unmethylated templates . After this amplification, highly methylated DNA sequences contain a higher number of CpG sites compared to unmethylated templates, which results in a different melting temperature that can be used in quantitative methylation detection . </Li> <Li> Ancient DNA methylation reconstruction, a method to reconstruct high - resolution DNA methylation from ancient DNA samples . The method is based on the natural degradation processes that occur in ancient DNA: with time, methylated cytosines are degraded into thymines, whereas unmethylated cytosines are degraded into uracils . This asymmetry in degradation signals was used to reconstruct the full methylation maps of the Neanderthal and the Denisovan </Li> </Ul> <Li> Mass spectrometry is a very sensitive and reliable analytical method to detect DNA methylation . MS in general is however not informative about the sequence context of the methylation, thus limited in studying the function of this DNA modification . </Li>

How can the binding of methyl groups imprinted genes