DNA Hydroxymethylation Analysis


DNA hydroxymethylation is a recently discovered type of DNA modification wherethe hydrogen atom at the C5 position in cytosine is replaced by a hydroxymethyl group. Although structurally similar to the 5-methylcytosine (5mC), the 5-hydroxymethylcytosine (5hmC) is much less abundant. The presence of 5hmC has been observed in the genomes of many different organisms, and it is a rich epigenetic marker in mammals. Moreover, its levels are the highest in the brain and other tissues of the central nervous system in mammals. 5hmC is produced by oxidation of 5mC through the Ten-Eleven Translocation (TET) family of methylcytosine dioxygenases and is recognized to be an intermediate step in the DNA demethylation pathway independent of replication. However, DNA methyltransferase (DNMT) enzymes are also thought to play a role in the production of 5hmC, suggesting that 5hmC may not only be considered as an intermediate in the 5mC demethylation process, but also as a significant epigenetic marker enriched within genomes. 5hmC and TETs play an important role in epigenetic reprogramming and regulation of tissue-specific gene expression and participate in normal development and pathogenesis of many diseases. Many techniques commonly utilized for the determination of classic DNA methylation (5mC) cannot distinguish between 5hmC and 5mC, and because of the low abundance of 5hmC, detection of 5hmC is technically more challenging than 5mC.

DNA hydroxymethylation (5hmC)

Figure 1. DNA hydroxymethylation (5hmC)

Creative BioMart offers a variety of DNA hydroxymethylation analysis services which can provide information on DNA hydroxymethylation at different levels, spanning from single site hydroxymethylation at specific locus of interest to genome-wide hydroxymethylation content. A comprehensive platform of epigenetic research at Creative BioMart meets all of your requirements in DNA hydroxymethylation analysis.

DNA Hydroxymethylation Analysis Services at Creative BioMart

Oxidative Bisulfite Sequencing Service

Oxidative bisulfite sequencing (oxBS-Seq) adds an additional oxidative step to bisulfite to distinguish 5hmC from 5mC. First, 5hmC in genomic DNA is oxidized to 5-formylcytosine (5fC). Unlike 5mC and 5hmC, 5fC is sensitive to bisulfite treatment, therefore bisulfite treatment of the oxidized DNA converts the 5fC to uracil (U).

Tet-Assisted Bisulfite Sequencing Service

Tet-assisted bisulfite sequencing (TAB-Seq), which utilizes the activity of the Ten-eleven translocation (TET) family protein to gradually oxidize the 5mC. This technique is based on bisulfite sequencing, detecting 5hmC at single-base resolution and quantifying its abundance at each site.

hMeDIP-based Service

Hydroxymethylated DNA immunoprecipitation (hMeDIP), which is modified from MeDIP, has become an invaluable tool for determining the relative abundance of 5hmC at specific loci or throughout the entire genome. hMeDIP involves immunoprecipitation using anti-5hmC antibodies and subsequent analysis by next-generation sequencing, qPCR, as well as microarray.

Global DNA 5hmC Quantification by LC-MS/MS

Liquid chromatography tandem mass spectrometry/mass spectrometry (LC-MS/MS) provides absolute measures of 5hmC content in genomic DNA. It is highly accurate and sensitive and is regarded as the ‘gold standard’ for global DNA hydroxymethylation measurement.

Global DNA 5hmC ELISA Quantification

The ELISA-based assay detects and quantifies 5hmC levels in genomic DNA from different sample types. This method is only suitable for the rough estimation of DNA hydroxymethylation. However, it enables higher throughput processing of samples with only a small amount of genomic DNA per well.

A group of well-trained and knowledgeable experts at Creative BioMart are dedicated to working with you to study DNA hydroxymethylation at a flexible and one-stop manner. We can help you with experimental design and perform your experiments as well as data analysis. Each of the techniques provided has its own pros and cons. Although you can choose the techniques you prefer, we can also help recommend. Moreover, we can provide a customized combination of services for comprehensive and in-depth DNA hydroxymethylation analysis. Our professional experts can help you design the optimal research protocol and eliminate all the hurdles in your program.

With more than 10 years of experience and skills, Creative BioMart is a leading company supplying a wide range of platforms for DNA hydroxymethylation analysis services to accelerate your epigenetic research within the shortest timeline. If you are interested in DNA hydroxymethylation analysis, please don’t hesitate to contact us, Creative BioMart is looking forward to working together with your attractive projects.

References
1. Branco M R.; et al. Uncovering the role of 5-hydroxymethylcytosine in the epigenome. Nature Reviews Genetics. 2011, 13(1): 7.
2. Richa R, Sinha R P. Hydroxymethylation of DNA: an epigenetic marker. Excli Journal. 2014, 13(5): 592-610.

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