Exclusive Highly-Specific Kits andAntibodies for DNA HydroxyMethylation studies
Jean-Jacques Goval, Irina Panteleeva, Géraldine Goens, Jan Hendrickx & Dominique Poncelet.
Diagenode sa, CHU, Tour GIGA B34, 3ème étage 1 Avenue de l’Hôpital, 4000 Liège, Sart-Tilman, Belgium

Introduction

Results

5-hydroxymethylcytosine (5-hmC) has been recently discovered in
mammalian DNA by two US groups (Kriaucionis & Heintz, Science, 2009
and Tahiliani et al. ,Science, 2009), however, its precise function has not
yet been elucidated. This cytosine modification results from the enzymaticconversion of 5-methylcytosine into 5-hydroxymethylcytosine by the TET
family of oxygenases. 5-hydroxymethylcytosine may represent a newpathway to demethylate DNA involving a repair mechanism convertinghmC to C and, as such open up entirely new perspectives in epigenetic
studies. Since its discovery in neuronal Purkinje, granule and ES cells,
studies of this new modified DNA base have been limited by the lack of high quality, validated tools and
5-hmC
5-mC
technologies that discriminate hydroxymethylation from methylation in regulating genome expression.
Obtaining a specific assay for 5-hmC is particularly important since standard methods (eg. bisulfitesequencing) cannot distinguish between these two types of methylation.

These affinity-based technologies appear to be the most powerful way to differentially and specificallyenrich 5-mC and 5-hmC sequences. Our monoclonal (mouse and rat) and polyclonal (rabbit) antibodies
for 5-hmC have been extensively validated using dotblots, immunofluorescence, hMeDIP, as well as
hMeDIP followed by next-gen sequencing.

Figure 2. Dotblot analysis of the Diagenode 5-hmC mouse monoclonal antibody
with the C, mC and hmC PCR controls.
200 to 2 ng (equivalent of 10 to 0.1 pmol of C-bases) of the hmC (1), mC (2) and C (3)PCR controls from the Diagenode “5-hmC, 5-mC & cytosine DNA Standard Pack”(Cat. No. AF-101-0020) were spotted on a membrane (Amersham Hybond-N+). The
membrane was incubated with 2 µg/ml of the mouse 5-hydroxymethylcytosine
monoclonal antibody (dilution 1:500). The membranes were exposed for 30
seconds.

5-mCc.

d.

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a.

b.

c.

c.

a. Add magnetic beads coated with antibody of interestb. Magnetic capture of Antibody - DNA complex
c. Washes
d. DNA isolation before qPCR

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Figure 1. Principle of the MeDIP and hMeDIP assays
Genomic DNA from cultured cells or tissues is prepared, sheared, and then denatured (1. and 2.). Immunoselection andimmunoprecipitation can take place using the antibody directed against 5 methylcytosine or 5-hydroxymethylcytosine
and antibody binding magnetic beads. After isolation and purification is performed, the IP’d DNA is ready for
subsequent analysis such as qPCR, amplification, hybridization on micro-arrays or next generation sequencing.

Principle of the Dual MeDIP assay
Dual MeDIP is a 2-step immunocapture approach developed by Diagenode to selectively enrichhydroxymethylated or methylated DNA. Genomic DNA is randomly sheared by sonication, thenimmunoprecipitated sequentially – first with an antibody that specifically recognizes 5-hmC, and thenwith an antibody-%+%)%'%%'(#&(%#&)%#&&

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c:D7Figure 4. Hydroxymethylated DNA IP
results obtained with our hMeDIP kit(Cat. No. AF-104-0016).

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^c:D7 Idhf-& Ed[Ykj'I\_'@f^'Hydroxymethylated DNA IP (hMeDIP)
assays were performed using theDiagenode hMeDIP kit. This kit includes:
the monoclonal rat antibody against
5-hydroxymethylcytosine, 5-hmC, 5mC& cytosine DNA standards Rat IgG (Cat.
No. AF-105-0025). 1 µg of mouse ES
cells DNA was spiked with each DNAstandard. Snrp70, Onecut1, Sfi1 andJph1 are hydroxymethylated locus fromthe mouse DNA sample.

In situ cellular staining on interphase ES cells indicates distinct 5-hmC staining, both in overallbrightness and in binding patterns, from that obtained with the Diagenode 5-mC antibody clone 33D3.
Figure 5. Indirect immunofluorescence results obtainedwith the antibody directed against 5-methylcytosine
and 5-hydroxymethylcytosine from Diagenode.
J1 ES cells were stained with 5-methylcytosine mousemonoclonal antibody (MAb), the monoclonal rat antibody
against 5-hydroxymethylcytosine and nuclei werecounterstained with DAPI.

Ordering information
Description

A methylated DNA IP (MagMeDIP)
was performed on the unboundfraction of the hydroxymethylated
DNA IP (hMeDIP) assays, using theDiagenode antibody directed against
5-hydroxymethylcytosineand5methylcytosine. IgG isotype antibody
from rat (Cat. No. AF-105-0025) wasused as a negative control.

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2. DNA Extraction andShearing using Bioruptor®

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b5-mC-%+%*"5-hmC

5-mC

Figure 3. Dual DNA IP results obtainedwith Diagenode’s hMeDIP kit andantibodies against methylated andhydroxymethylated C.

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3. Immunoprecipitation, washes and DNA isolation

5-hmC

5-mC:kWbC[:?F&'%&%%]b

1. Cell collection and lysis

Our new hMeDIP kit includes a hmc antibody, and positive (ie, fully hydroxy-methylated) and negative(ie, both unmethylated and fullymethylated) DNA controls.

]bWe have developed hMeDIP, MeDIP and dual MeDIP kits for the differential study of the functions of
5-hm, 5-m, and unmodified Cytosines.

*"Methods

Cat. No.

Format5-hmC monoclonal antibody (rat)MAb-633HMC-050/100

50 μg or 100 μg

5-hmC monoclonal antibody (mouse)

MAb-31HMC-050/100

50 μg or 100 μg

5-hmC polyclonal antibody (rabbit)

CS-HMC-100

100 μl

5-mC monoclonal antibody clone 33D3 (mouse)

Mab-081-100/500

100 μg or 500 μghMeDIP kit x 16 (rat)(for mouse and rabbit antibodies also available)

AF-104-0016

16 rxns

F. Santos June 2010

Our data are consistent with distinct roles of 5-hmC and 5-mC; our new kits and antibody open thedoor to novel epigenetics studies, and to clarifying the role of 5-hmC in differentiation, displacing
methyl-binding proteins, regulating DNA repair, recruiting chromatin modifiers, and other importantfunctions.

Conclusions
In conclusion, we present the most complete range of antibodies against 5-hydroxymethylcytosine thathave been demonstrated to be highly specific (no cross-reactivity with C– or mC– containing fragments)
with our « dual MeDIP » and hMeDIP assays as well as dotblots and that outperform other antibodies
on the market. We have developed an efficient test system that can be used to assess the performanceof the antibody, and to compare hydroxymethylated and standard methylated DNA distributions. We
expect that our assays and antibodies will represent the first tools for evaluating the role of differential
methylation in the genome. These tools should be relevant and useful in any cell type.

For more information, please contact Dominique.poncelet@diagenode.com • For Europe, contact Orders@diagenode.com • For the US and Canada, contact Orders.NA@diagenode.com