Table 1.
Representative list of commonly used methodologies for analyzing DNA methylation1
| Description | Pros | Cons | References | |
|---|---|---|---|---|
| Global | ||||
| Liquid chromatography tandem mass spectrometry | Digestion/hydrolysis of genomic DNA to nucleosides and fractionation by LC-MS | Highly sensitive, quantitative, accurate, and reproducible | Requires costly instrumentation; requires ~ 1 μg amounts of DNA | Shellnut et al. (137) |
| HPLC | Digestion/hydrolysis of genomic DNA to nucleotides or bases and fractionation on HPLC | Highly quantitative, accurate, and reproducible | Requires costly instrumentation; requires μg amounts of DNA | Armstrong et al. (186) |
| Methyl acceptance | Global methylation determined by level of in vitro methylation of genomic DNA by SssI methylase | Technical simplicity | Lacks sensitivity; imprecise; weak reproducibility | Balaghi and Wagner (187) |
| Gene specific | ||||
| Methyl-sensitive restriction enzyme digestion | Assesses methylation at specific restriction sites using Southern blotting | Standard molecular biology techniques | Limited to analysis of individual restriction sites; not strictly quantitative | Wolf and Migeon (188) |
| Methylation-specific polymerase chain reaction | Specifically amplifies methylated genomic DNA sequences after sodium bisulfite treatment of genomic DNA | Sensitivity; technical simplicity | Not quantitative; limited sequence coverage | Herman et al. (189) |
| Pyrosequencing | Determines level of methylation at specific CpG sites | Highly quantitative; single-nucleotide resolution; technically straightforward | Requires costly instrumentation; restricted sequence coverage | Tost and Gut (190) |
| Microarray | Large numbers of individual CpG sites from specific genes are assayed using standard microarray equipment after bisulfite treatment; 1500 > 450K sites per sample | Quantitative; specific; technically straightforward; large number of CpGs at a time | Requires costly instrumentation; requires ~0.5 μg DNA | Bibikova et al. (191), Christensen et al. (131), Fernandez et al. (14) |
| Sodium bisulfite genomic sequencing | Determines methylation status of each cytosine in a region of interest within a single DNA molecule | Single-nucleotide resolution; reveals DNA methylation patterns of individual DNA molecules | Labor intensive; time-consuming; restricted sequence coverage | Clark et al. (192) |
| Genome-wide profiling | ||||
| MethylC-seq | Determines methylation status of each cytosine in the genome after chemical conversion of unmethylated cytosines to uracils in genomic DNA | Single-nucleotide resolution; full genome coverage; the most comprehensive method for genome-wide profiling of DNA methylation | Requires sequencing of full genome at ~15-fold coverage; cost | Lister et al. (45) |
| meDIP/meDNA pull-down/MIRA | Uses antimethylated cytosine antibody or methylated DNA-binding protein to enrich for methylated DNA | Successfully used in a wide variety of published studies; technically accessible | Low resolution, not full genome coverage; efficiency dependent on CpG density | Weber et al. (193), Nair et al. (194), Rauch et al. (195) |
| RRBS | A size-selected portion of the genome is isolated after restriction enzyme digestion and sequenced after sodium bisulfite treatment | Single-nucleotide resolution | Biased toward analyzing CpG islands; lacks full genome coverage | Gu et al. (196) |
| CHARM | Uses restriction enzyme that specifically cleaves methylated DNA | Quantitative | Lacks full genome coverage | Irizarry et al. (197) |
| HELP | Compares the relative representation of MspI and HpaII fragments at individual loci | Assays both CpG islands and non-CpG island sites; quantitative | Lacks full genome coverage | Khulan et al. (198) |
| MSCC | Assesses methylation status at all recognition sites of a methylation-sensitive restriction enzyme | High resolution; not restricted to CpG islands | Not strictly quantitative; indirect assessment of methylation by detecting unmethylated CpG sites; lacks full genome coverage | Ball et al. (199) |
For sodium bisulfite conversion used in methylation-specific polymerase chain reaction, pyrosequencing, sodium bisulfite genomic sequencing, full genome sequencing, RRBS, etc., all cytosines except methylated cytosines in genomic DNA are chemically converted uracil by sodium bisulfite; methylated cytosines are resistant to conversion and can be detected by various methods. (This table is not a comprehensive list of methods used to assay DNA methylation.) CHARM, comprehensive high-throughput arrays for relative methylation; HELP, HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction; HPLC, high-performance liquid chromatography; LC-MS, liquid chromatography-mass spectrometry; meDIP, methylated DNA immunoprecipitation; meDNA, methylated DNA; MIRA, methylated CpG island recovery assay; MSCC, methylation-sensitive cut counting; RRBS, reduced representation bisulfite sequencing.