In mammalian genomes, 5-mC can be enzymatically oxidized to 5-hmC (5-hydroxymethylcytosine). There is intense scientific curiosity on identifying the epigenetic mechanisms and functions of the 5-hmC. This modification is suggested to be an intermediate between methylation and demethylation of the genome. Occupancy of 5-hmC correlates with inactive or non-productive promoters (1). 5-hmC pattern is different from 5-methylcytosine (5-mC) in many instances (2). Recently, a few analytical methods like chromatography coupled to mass spectroscopy and 5-hmC DNA immunoprecipitation have been developed to quantitate and analyze 5-hmC. However, all the methods fall short of acheiving quantitative site specific resolution.
FAQs for 5-hmC Detection & Analysis
- Does the T4-BGT show any site preference?
- Do I need to do extra purification steps for a mammalian DNA prep in order to use it in a glucosylation reaction?
- How do I know if the glucosylation of 5-hmC in my DNA is complete?
- How much control DNA should I use in my qPCR?
- Has NEB used any enzymes in Chromatin Conformation Capture techniques, such as 3C or HiC?
- What is the shelf life of the reagents supplied?
- What products does NEB offer for the study of Epigenetics?
Protocols for 5-hmC Detection & Analysis
- Cross-linking of IgG to Protein A or G Beads
- Double Digest Protocol with Standard Restriction Enzymes
- Immunoprecipitation using Protein A/G Magnetic Beads
- mRNA Isolation using Streptavidin Magnetic Beads (NEB #S1420 and NEB #S1421)
- Optimizing Restriction Endonuclease Reactions
- Phage Display: Solution-phase Panning with Affinity Bead Capture
- Protocol for Glucosylation and digestion of Genomic DNA using AbaSI (#R0665)
- Purification of IgG using Protein A/G Magnetic Beads
- Reaction Protocol for EpiMark® 5-hmC and 5-mC Analysis Kit (E3317)
Application Notes 5-hmC Detection & Analysis
- Bhattacharyya, S., Yu, Y., Suzuki, M., Campbell, N., Mazdo, J., Vasanthakumar, A., et al. 2013. Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer Nucleic Acids Research. , PubMedID: , DOI: doi:10.1093/nar/gkt601
Publications related to 5-hmC Detection & Analysis
EpiMark® 5-hmC and 5-mC Analysis Kit Overview
Follow NEB Product Development Scientist, Romas Vaisvila, Ph.D., as he demonstrates the EpiMark® 5-mc and 5-hmc Analysis Kit for locus specific identification and quantification of 5-hmc in this protocol "High Sensitivity 5-hmc detection in Balb/C Brain Tissue".
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.