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DNA Methylation Table

There are several approaches for studying DNA methylation, based on enzymatic conversion, pretreating genomic DNA with sodium bisulfite, restriction enzymes or methylated DNA-binding affinity matrix.

Method Description Advantages Disadvantages
NEBNext® Enzymatic Methyl-seq (EM-seq™)

EM-seq is an enzymatic alternative to bisulfite conversion for detection of 5mC and 5hmC, using a two-step method:

  1. Protection of 5mC and 5hmC from deamination, by TET2 and an Oxidation Enhancer;
  2. APOBEC deamination of unprotected cytosines, converting them to uracils, while the protected 5mC and 5hmC bases are not deaminated.

Cystosine methylation information at single-nucleotide resolution is achieved by sequence data comparison between the reference genome and EM-seq-treated DNA.
  • Gentle enzyme-based conversion treatment minimizes damage to DNA
  • Sensitive detection of 5mC and 5hmC
  • More uniform GC coverage
  • Input amounts as low as 10 ng
  • Compatible with automation
  • Compatible with bioinformatic analysis pipelines used for bisulfite sequencing
  • Like bisulfite conversion, EM-seq detects both 5mC and 5hmC but does not distinguish between them
Sodium Bisulfite Conversion Treatment of denatured DNA (i.e., single-stranded DNA) with sodium bisulfite leads to deamination of unmethylated cytosine residues to uracil, leaving 5-mC intact. The uracils are amplified as thymines, and 5-mC residues are amplified as cytosines in PCR. Comparison of sequence information between the reference genome and bisulfite-treated DNA can provide single-nucleotide resolution information about cytosine methylation patterns.
  • Resolution at the nucleotide level
  • Works on 5-mC-containing DNA
  • Automated analysis
  • Requires micrograms of DNA input
  • Harsh chemical treatment of DNA can lead to its damage
  • Potentially incomplete conversion of DNA
  • Cannot distinguish between 5-mC and 5-hmC
  • Multi-step protocol
Sequence-Specific Enzyme Digestion Restriction enzymes are used to generate DNA fragments for methylation analysis. Some restriction enzymes are methylation-sensitive (i.e., digestion is impaired or blocked by methylated DNA). When used in conjunction with an isoschizomer that has the same recognition site but is methylation insensitive, information about methylation status can be obtained. Additionally, the use of methylation-dependent restriction enzymes (i.e., requires methylated DNA for cleavage to occur) can be used to fragment DNA for sequencing analysis.
  • High enzyme turnover
  • Well-studied
  • Easy-to-use
  • Availability of recombinant enzymes
  • Determination of methylation status is limited by the enzyme recognition site
  • Overnight protocols
  • Lower throughput
Methylated DNA Immunoprecipitation Fragmented genomic DNA (restriction enzyme digestion or sonication) is denatured and immunoprecipitated with antibodies specific for 5-mC. The enriched DNA fragments can be analyzed by PCR for locus-specific studies or by microarrays (MeDIP-chip) and massively parallel sequencing (MeDIP-seq) for whole genome studies.
  • Relatively fast
  • Compatible with array-based analysis
  • Applicable for high-throughput sequencing
  • Dependent on antibody specificity
  • May require more than one 5-mC for antibody binding
  • Requires DNA denaturation
  • Resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design
  • Data from repeat sequences may be overrepresented
Methylated DNA-Binding Proteins Instead of relying on antibodies for DNA enrichment, affinity-based assays use proteins that specifically bind methylated or unmethylated CpG sites in fragmented genomic DNA (restriction enzyme digestion or sonication). The enriched DNA fragments can be analyzed by PCR for locus-specific studies or by microarrays and massively parallel sequencing for whole genome studies.
  • Well-studied
  • Does not require denaturation
  • Compatible with array-based analysis
  • Applicable for high-throughput sequencing
  • May require high DNA input
  • May require a long protocol
  • Requires salt elutions
  • Does not give single base methylation resolution data