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DNA Methylation Variants (5-mC, 5-hmC, 5-fC, 5-caC & 3-mC)

The field of DNA methylation analysis has expanded recently with the identification of multiple cytosine variants. Traditional DNA methylation involves the transfer of a methyl group to the carbon 5 position of cytosine to produce 5-methylcytosine (5-mC). However, research has shown that the Tet family of cytosine oxygenase enzymes are involved in oxidizing 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). To learn more about each of these DNA methylation variants or to see the list of available products Active Motif offers for the analysis of the individual DNA modifications, please click on the links below.

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Structure of 5-Methylcytosine (5-mC)

5-Methylcytosine (5-mC)

5-Methylcytosine is the DNA modification that results from the transfer of a methyl group from S-adenosyl methionine (also known as AdoMet or SAM) to the carbon 5 position of a cytosine residue. This transfer is catalyzed by DNA methyltransferase enzymes (DNMTs)1. 5-Methylcytosine is the most common and widely studied form of DNA methylation. It usually occurs within CpG dinucleotide motifs, although non-CpG methylation has been identified in embryonic stem cells2. Active Motif offers a variety of antibodies and assays specifically designed to aid researchers in the analysis of 5-methylcytosine DNA methylation.

For methylated DNA enrichment of fragments containing 5-methylcytosine, Active Motif offers the antibody-based MeDIP Assay in which an antibody specific for 5-methylcytosine is used to bind and immunoprecipitate methylated DNA fragments. This reduces sample complexity, leaving you with DNA specifically enriched for 5-mC. Active Motif also offers an alternative method for enrichment that uses Methyl-CpG binding proteins (MBD) to specifically bind DNA fragments containing 5-methylcytosine within a CpG dinucleotide. Active Motif's MethylCollector™ Ultra Kit uses a combination of MBD2b and MBD3L1 to enhance the binding and enrichment of 5-mC DNA.

Researchers interested in studying the DNA methyltransferase enzymes, DNMT1, DNMT3a and DNMT3b, that are responsible for transferring the methyl group to the fifth carbon of cytosine to produce 5-methylcytosine should use the DNMT Activity / Inhibition Assay. This non-radioactive assay can screen DNMT enzymes or nuclear extract samples for their ability to methylate the oligonucleotide substrate coated on the wells of a 96-well plate. The colorimetric assay is easily quantified by spectrophotometry and is ideally suited to screen for inhibitors of DNMTs or to evaluate changes in the DNMT activity across samples.

A feature of various human diseases including cancer is genome-wide alterations in DNA methylation content. For researchers interested in assaying global DNA methylation, Active Motif's Global DNA Methylation Assay – LINE-1 is a convenient ELISA-based assay for the detection and quantification of global 5-mC levels in human genomic DNA. The 96-well assay utilizes a unique hybridization approach that quantitates 5-mC levels of Long Interspersed Nucleotide Element 1 (LINE-1) repeats as a surrogate measure for global DNA methylation. This hybridization approach offers better specificity and reproducibility than methods that use passive adsorption for DNA capture. The assay is ideally suited for screening relative changes in 5-mC levels across multiple samples resulting from variables such as treatment conditions, patient history and clinical prognosis.

If you are simply looking for positive controls to assist in validating your research, Active Motif offers both a Methylated DNA Standard Kit and Fully Methylated Jurkat DNA. The Fully Methylated Jurkat DNA has been enzymatically treated to ensure 5-methylcytosine DNA methylation of all CpG dinucleotides. This methylated DNA serves as an excellent control for our MethylCollector™ Ultra assay, or any experiment involving the analysis of CpG methylation. The Methylated DNA Standard Kit is composed of three different DNA standards: unmethylated standard, 5-methylcytosine standard and 5-hydroxymethylcytosine standard. The kit contains all three DNAs and corresponding sequence-specific PCR primers to analyze each DNA modification individually. These controls can be used for dot blot analysis or as spiked controls in enrichment experiments.

Since recent studies have shown that the Tet family of Fe(II) and 2-oxoglutarate-dependent cytosine oxygenase enzymes are responsible for the conversion of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine, Active Motif also offers a Recombinant Tet1 protein for the analysis of this conversion process, or to study the conversion of 5-methylcytosine to the various DNA methylation variants3,4,5.

For more complex analysis of 5-methylcytosine DNA methylation that may surpass the capabilities of your research laboratory, or the availability of resources with the required level of skill and expertise in this area, Active Motif also offers Epigenetic Services for the analysis of 5-methylcytosine. Available services include MeDIP-Seq and bisulfite conversion, to name a few. Please click on the link, Epigenetic Services, for more complete information.

Structure of 5-Hydroxymethylcytosine (5-hmC)

5-Hydroxymethylcytosine (5-hmC)

5-Hydroxymethylcytosine is a DNA methylation modification that occurs as a result of enzymatic oxidation of 5-methylcytosine (5-mC) by the Tet family of iron-dependent deoxygenases3. 5-Hydroxymethylcytosine can be found in elevated amounts in certain mammalian tissues, such as mouse Purkinje cells and granule neurons6. Alternatively, 5-hmC may be produced by the addition of formaldehyde to DNA cytosines by DNMT proteins7.

While a variety of techniques exist to study DNA methylation, most of these techniques were developed to distinguish 5-methylcytosine from unmodified cytosine. Evaluations of these methods in the context of 5-hydroxymethylcytosine reveal that many are not suited for the analysis of 5-hmC. Bisulfite sequencing, for example, cannot distinguish between 5-mC and 5-hmC, just as methyl-binding protein methods (e.g. methylated CpG island recovery assay, MIRA) will only recognize 5-mC DNA8. To better understand the function of 5-hydroxymethylcytosine in the mammalian genome, new tools and techniques need to be employed.

Methylated DNA enrichment techniques specific for 5-hydroxymethylcytosine offer one approach to specifically isolate and study hydroxymethylation. Hydroxymethylated DNA Immunoprecipitation (hMeDIP) is an antibody-based enrichment technique where an antibody specific for the 5-hydroxymethylcytosine residue is used to bind DNA fragments containing 5-hydroxymethylcytosine. The antibody/DNA complex is then pulled down from the rest of the genomic DNA, resulting in a final elution that is highly enriched for 5-hydroxymethylcytosine DNA. Active Motif's hMeDIP Kit is designed for this exact purpose and has been optimized for use with double-stranded DNA.

An alternative method for enrichment of hydroxymethylated DNA is to modify the 5-hydroxymethylcytosine residue such that it becomes distinct from 5-methylcytosine, thereby allowing for enrichment of DNA fragments containing 5-hydroxymethylcytosine. Active Motif's Hydroxymethyl Collector™ Kit was developed to utilize the properties of the β-Glucosyltransferase enzyme to transfer a modified glucose moiety to 5-hmC residues in double-stranded DNA9. Using the chemical properties of the modified glucose, a biotin conjugate can then be attached which enables enrichment of hydroxymethylated DNA with the use of streptavidin magnetic beads10. This covalent labeling of 5-hmC ensures accurate capture of DNA fragments containing 5-hydroxymethylcytosine and is an efficient method to separate hydroxymethylation specifically from other cytosine modifications.

The use of methylation-sensitive restriction enzymes is another way to discriminate between 5-hmC and 5-mC. To date, there is only one enzyme, the PvuRts1 I restriction enzyme, capable of directly differentiating these two forms of DNA methylation. The PvuRts1 I enzyme is specific to 5-hmC DNA and will not digest 5-methylcytosine residues or unmethylated DNA11.

Another way to distinguish 5-mC and 5-hmC methylation is targeted modification of the 5-hydroxymethylcytosine residue. For this purpose, the β-Glucosyltransferase enzyme is able to transfer a glucose moiety from uridine diphosphoglucose (UDP-Glucose) to the 5-hydroxymethylcytosine residue in double-stranded DNA, creating glucosyl-5-hmC DNA. Glucosylated DNA can be quantified or used to differentiate 5-hmC and 5-mC DNA using glucosyl-sensitive restriction enzymes9.

For researchers interested in studying the conversion of 5-methylcytosine into 5-hydroxymethylcytosine, the Recombinant Tet1 protein is available. Continued activity of the Tet1 protein on 5-hydroxymethylcytosine will initiate its conversion to 5-formylcytosine and 5-carboxylcytosine.

For researchers in need of positive control DNA, Active Motif has also developed our Methylated DNA Standard Kit. The Methylated DNA Standard Kit contains three recombinant standards derived from the APC gene promoter. Each standard is 338 base pairs and contains 122 cytosine residues. One standard is completely unmethylated, while another standard is fully 5-methylcytosine methylated and the last standard is fully 5-hydroxymethylcytosine methylated. PCR primers for the APC gene are included with the Methylated DNA Standard Kit. The Methylated DNA Standards can be used for dot blot analysis, or as spike controls in methylation assays.

If your lab is not able to perform 5-hydroxymethylcytosine DNA analysis, consider using Active Motif's Epigenetic Services to provide the expertise, knowledge, and experience to perform the experiments you need. Our services for the analysis of 5-hydroxymethylcytosine include hMeDIP-Seq and hMeDIP-chip. Please click on the link, Epigenetic Services, for more details.

Structure of 5-Formylcytosine (5-fC)

5-Formylcytosine (5-fC)

Recent publications have demonstrated that 5-formylcytosine is one of the DNA variants that is produced when Tet enzymes act on 5-hydroxymethylcytosine4,5. Further oxidation of 5-formylcytosine by the Tet enzyme will results in conversion to 5-carboxylcytosine. It is believed that the oxidation of 5-methylcytosine through the various DNA methylation variants represents a mechanism of DNA demethylation, and that this demethylation pathway has a function during development and germ cell programming. 5-Formylcytosine is present in mouse embryonic stem (ES) cells and major mouse organs4. This DNA modification also appears in the paternal pronucleus post-fertilization, concomitant with the disappearance of 5-methylcytosine, suggesting its involvement in the DNA demethylation process12. To help researchers elucidate the function of 5-formylcytosine, Active Motif offers antibodies for the study of 5-fC.

Structure of 5-Carboxylcytosine (5-caC)

5-Carboxylcytosine (5-caC)

5-Carboxylcytosine has been identified as one of the DNA methylation variants that is produced when Tet enzymes oxidize 5-hydroxymethylcytosine and, subsequently 5-formylcytosine4,5. It is believed that the oxidation of 5-methylcytosine through to 5-carboxylcytosine represents a mechanism of DNA demethylation, and that this demethylation pathway has a function during development and germ cell programming. It has been suggested that 5-caC is excised from genomic DNA by thymine DNA glycosylase (TDG), which returns the cytosine residue back to its unmodified state5. 5-Carboxylcytosine has been identified in mouse embryonic stem (ES) cells4. This DNA modification appears in the paternal pronucleus post-fertilization, concomitant with the disappearance of 5-methylcytosine, further lending support that this variant is part of a DNA demethylation pathway12. To help researchers elucidate the function of 5-carboxylcytosine, Active Motif offers antibodies for the study of 5-caC. Additionally, Active Motif also offers a 5-Carboxylcytosine DNA Standard Kit that includes two double-stranded DNA oligonucleotides: unmodified DNA standard and 5-carboxylcytosine DNA standard, which can be used as negative and positive controls, respectively, in the analysis of 5-carboxylcytosine.

Structure of 3-Methylcytosine (3-fC)

3-Methylcytosine (3-mC)

3-Methylcytosine is unlike the other DNA methylation variants in that this modification is not associated with the oxidative pathway of the Tet family of proteins. Instead, 3-methylcytosine is an adduct created via spontaneous exposure of the nitrogen-three base of cytosine to endogenous S-adenosyl methionine. 3-Methylcytosine is mutagenic and is repaired through base excision repair (BER) in humans or by dealkylation via human homologues of the E. coli AlkB protein. Cells with a loss of ALKBH3 show an increase in 3-methylcytosine and a reduction in cell proliferation13. For researchers interested in this area of research, Active Motif offers antibodies specific for 3-Methylcytosine.


  1. Latham, T., Gilbert, N. & Ramsahoye, B. (2008) Cell Tissue Res 331, 31-55.
  2. Ramsahoye, B. et al. (2000) PNAS 97, 5237-5242.
  3. Tahiliani, M, et al. (2009) Science 324, 930-935.
  4. Ito, S. et al. (2011) Science 333, 1300-1303.
  5. He, Y.F. et al. (2011) Science 333, 1303-1307.
  6. Kriaucionis, S. and Heintz, N. (2009) Science 324, 929-930.
  7. Liutkeviciute, Z. et al. (2009) Nat. Chem. Biol. 5, 400-402.
  8. Jin, S.G. et al. (2010) Nucleic Acids Res doi:10.1093/nar/gkq223.
  9. Szwagierczak, A. et al. (2010) Nucleic Acids Res 38, e181.
  10. Song, C.X. et al. (2011) Nature Biotechnology 29, 68-72.
  11. Szwagierczak, A. et al. (2011) Nucleic Acids Res 39, 5149-5156.
  12. Inoue, A. et al. (2011) Cell Research 21, 1670-1676.
  13. Dango, S. et al. (2011) Mol Cell 44, 373-384.