Methods to analyze DNA methylation, DNA demethylation, and their functional effects are critical to epigenetics researchers. Methylation profiles of epigenome are used for disease identification and for research and therapeutic development. Analysis can be gene-specific or global depending on downstream applications (1).
Downstream methylation detection methods include end-point PCR, real-time PCR, primer extension, single-stranded conformational polymorphism assays, blotting, microarray analysis and sequencing. Choosing which method(s) to use largely depends on the experimental sample size and the goals of the experiments.
- Tao Zuo et al. 2009, Epigenomics 1(2) 331-345. PMID: 20526417
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Epigenetics - Expanding on Genomic Foundations
- Epigenetics Brochure
- EpiMark® Methylated DNA Enrichment Kit Troubleshooting Guide
- Woodcock, C.B.l, Yu, D., Hajian, T., Li, J., Huang, Y., Dai, N., Corrêa, I.R., Jr., Wu, T., Vedadi, M., Zhang, X., Cheng, X (2019) Human MettL3 MettL14 complex is a sequence specific DNA adenine methyltransferase active on single strand and unpaired DNA in vitro Cell Discov ; 5:63, PubMedID: 31885874, DOI: 10.1038/s41421-019-0136-4
- Pirone-Davies, C., Hoffmann, M., Roberts, R.J., Muruvanda, T., Timme, R.E., Strain, E., Luo, Y., Payne, J., Luong, K., Song, Y., Tsai, Y.C., Boitano, M., Clark, T.A., Korlach, J., Evans, P.S., Allard, M.W. (2015) Genome-Wide Methylation Patterns in Salmonella enterica Subsp. enterica Serovars PLoS One; 10(4), e0123639. PubMedID: 25860355
- Pais, J.E., Dai, N., Tamanaha, E., Vaisvila, R., Fomenkov, A.I., Bitinaite, J., Sun, Z., Guan, S., Correa, I.R. Jr., Noren, C.J., Cheng, X., Roberts, R.J., Zheng, Y. and Saleh, L. (2015) Biochemical characterization of a Naegleria TET-like oxygenase and its application in single molecule sequencing of 5-methylcytosine Proc Natl Acad Sci U S A; 112(14), 4315-4321. PubMedID: 25831492
- 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), 205-212. 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), 1335-50. PubMedID: 22722369, DOI: 10.1038/nprot.2012.071
- Bisulfite conversion, which provides single nucleotide, high resolution data for whole genomes or single locus methylation studies.
- Differential enzymatic cleavage, which uses methylation, or dually 5-mC and 5-hmC dependent enzymes, paired with non-sensitive isoschizomers to asses DNA methylation or DNA hydroxymethylation.
- High-Performance Liquid Chromatography (HPLC)
- Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry(MALDI-TOF MS)
- Methylated DNA-ELISA
- Affinity-based methods, which use methylated DNA binding proteins or antibodies for enrichment.
- Enzymatic DNA methyltransferase assay
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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.