Methylation of DNA is the most studied epigenetic modification and the earliest methods involved chromatography techniques and nearest-neighbor analysis. Following the discovery of CpG methylation in mammals, important new methods employing isoschizomers HpaII (NEB #R0171) and MspI (NEB #R0106) were utilized. The former does not digest if the internal CpG is methylated, where as the later cleaves the DNA. These enzymes are used in conjunction with PCR amplification with flanking primers or with Southern blot analysis (1). Most restriction enzymes are sensitive to the DNA methylation state of their recognition site where cleavage can be blocked or impaired when a particular base is modified. A class of restriction enzyme defies this rule. They are indeed dependent on methylation for cleavage to occur as exemplified by the MspJI family. This property is exploited to probe the methylation state of a genome (2). Choose the tab below to learn more about methylation profile methods that utilize restriction enzymes.
While restriction enzyme methods are often easy to use, the data is limited by the presence of enzyme recognition site(s) in the sequence of interest. However, a recent application of a restriction enzyme, particularly MspI (NEB #R0106), in reduced representation bisulphite sequencing (RRBS) has gained prominence due to the procedure’s enrichment of CpG island methylation analysis (3). Furthermore, there is renewed interest in using restriction enzyme based methods in identification, detection and quantification of hydroxymethylated from methylated DNA. Hydroxymethylated DNA is not differentiated from methylated DNA during bisulfite conversion and sequencing (4).
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- What's the difference between DpnI, DpnII, MboI, and Sau3AI?
- Does McrBC cut hemi-methylated DNA?
- Does McrBC produce blunt or sticky ends?
- Why does my McrBC cleaved DNA smear when run on an agarose gel?
- Will DpnI cleave hemimethylated DNA?
- Has NEB used any enzymes in Chromatin Conformation Capture techniques, such as 3C or HiC?
- Double Digest Protocol with Standard Restriction Enzymes
- Optimizing Restriction Endonuclease Reactions
- Protocol for Digestion Prior to droplet digital PCR (ddPCR)
- Protocol for Direct Digestion of gDNA during droplet digital PCR (ddPCR)
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using FspEI
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using LpnPI (R0663)
- Protocol for generating 32 bp fragments from modified CpG sites in genomic DNA using MspJI
- Marx V. 2016. Genetics: profiling DNA methylation and beyond Nature Methods. 13, PubMedID: , DOI: 10.1038/nmeth.3736
- Hughes J.R., Roberts N., McGowan S., Hay D., Giannoulatou E., Lynch M., De Gobbi M., Taylor S., Gibbons R., Higgs D.R. 2014. Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment Nat Genet. 46 (2), PubMedID: 24413732, DOI: doi:10.1038/ng.2871
- Sexton T, Kurukuti S, Mitchell JA, Umlauf D, Nagano T, Fraser P 2012. Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip Nat Protoc. 7(7), PubMedID: 22722369, DOI: 10.1038/nprot.2012.071
There are numerous other methods that utilize restriction enzymes for methylation profiling. These include:
- Methylation-Sensitive Cut Counting Assay (MSCC) involves analyzing untreated and bisulfite treated DNA by assessing differential migration of single-stranded DNA containing the CpG sites of interest through non-denaturing gels. The C to T content will vary with methylation status (1,2).
- Methylation specific PCR (MSP) involves analyzing untreated and bisulfite treated DNA using two sets of PCR primer pairs that target the unaltered, methylated sequence and the converted, unmethylated sequence (3,4).
- Quantitative Analysis by methylation-sensitive PCR (qAMP) involves using methylation sensitive enzymes to fragment genomic DNA for quantitative analysis by real-time PCR (5).
- Restriction Landmark Genome Scanning (RLGS) is a method that uses 2-dimensional gel electrophoresis to separate DNA fragments generated with methylation-sensitive restriction enzymes (6,7).
- Combined Bisulfite Restriction Analysis (COBRA) involves digesting PCR amplicons from untreated, and bisulfite treated DNA with methylation-sensitive or -insensitive restriction enzymes. The resulting DNA fragments are electroblotted, hybridized to radiolabeled oligonucleotides and quantitated by densiometry (8).
- High-Throughput Genome-wide methylation profiling and analysis, can be accomplished by CpG Island microarrays. CpG islands are targeted using oligonucleotide adaptors. These are prepared by two rounds of a combination of methylation-sensitive and methylation-insensitive nuclease digests, PCR amplicons are fluorochrome labeling, for microarray of sample and control genomic DNAs (9).
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If all cells are created from the same genetic material, why are there so many different cell types? Listen to Sriharsa Pradhan, Senior Scientist, RNA Biology at NEB, as he describes how DNA is methylated and how this affects the path of reading the DNA code the same way an obstruction would derail a train off its tracks.