This study was supported in part by the National Institutes of Health (GM071440 to C.H. and NS051630/MH076090/MH078972 to PJ).

1. Genomic DNA Fragmentation
Fragment genomic DNA using sonication to a desired size range suited for the genome-wide sequencing platform. (We usually sonicate to ~300 bp.) Verify the size distribution of the fragmented genomic DNA on 1% agarose gel (Figure 1).
2. DNA Preparation
Determine the starting DNA amounts based on the abundance of 5-hmC in genomic DNA. Since 5-hmC levels vary significantly in different tissue types, starting DNA amounts depend on the 5-hmC levels of the samples. Please refer to Table 1 for examples.
3. &beta;-GT Catalyzed Reaction (Glucose Transfer Reaction)
<ol>
 <li>Mix by pipetting the mixture as detailed in Table 2 and incubate in a 37 &deg;C water bath for 1 hr.</li>
 <li>After incubation, clean up the reaction with QIAquick Nucleotide Removal Kit, using 10 &mu;g of DNA per column. Elute with 30 &mu;l water per column and combine.</li>
</ol>
4. Biotinylation Reaction (Click Chemistry)
<ol>
 <li>Add DBCO-S-S-PEG3-Biotin conjugate working solution (1 mM) in the eluted DNA solution (from step 3) to a final concentration of 150 &mu;M (i.e., 5 &mu;l of working solution per 30 &mu;l DNA solution).</li>
 <li>Mix by pipetting and incubate in a 37 &deg;C water bath for 2 hr.</li>
 <li>Clean up the reaction with QIAquick Nucleotide Removal Kit. The ideal total elution volume is 100 &mu;l.</li>
 <li>Quantify the recovered DNA amount using microliter scale spectrophotometer (e.g., NanoDrop).</li>
</ol>
5. Capture of 5-hmC-containing DNA
<ol>
 <li>Wash 50 &mu;l of Dynabeads MyOne Streptavidin C1 3 times with 1 ml of 1X B&amp;W buffer according to the manufacturer's instructions. Separate the beads with a magnetic stand.</li>
 <li>Add equal volume of 2X B&amp;W buffer to the recovered biotinylated DNA (100 ul) to the washed beads.</li>
 <li>Incubate for 15 min at room temperature with gentle rotation on a rotator.</li>
 <li>Separate the beads with a magnetic stand and wash the beads 3 times with 1 ml of 1X B&amp;W buffer.</li>
 <li>Elute the DNA by incubating the beads in 100 &mu;l of freshly prepared 50 mM DTT for 2 hr at room temperature with gentle rotation on a rotator.</li>
 <li>Separate the beads with a magnetic stand. Aspirate the eluent and load onto a Micro Bio-Spin 6 Column according to the manufacture instruction to remove the DTT. The target DNA is in the solution now.</li>
 <li>Purify the eluted DNA from the previous step by Qiagen MinElute PCR Purification Kit and elute DNA in 10 &mu;l of EB buffer. Quantify DNA using Qubit Fluorometer, or NanoDrop if concentration is higher than 20 ng/ul. The DNA is ready for downstream genome-wide sequencing library preparation.</li>
</ol>
6. Representative Results
If the quality of genomic DNA is high, typical recovery yields after the &beta;-GT and biotinylation reactions are ~60-70%. However, the capture efficiency vary significantly with different tissue types depending on the 5-hmC levels of the samples. Typically, the capture efficiency for brain genomic DNA is ~4-9%, and in some extreme cases, the efficiency may reach up to 12%. For ES cells, the average capture efficiency is ~2-4%, in contrast to ~0.5% for neural stem cells. The lowest efficiency seen so far was for genomic DNA from cancer cells. All enriched DNA is ready for standard next-generation library preparation protocols. In addition, the captured DNA can also be used as template for real-time PCR to detect the enrichment of some fragments compared to the input DNA, if the related primers are available.
<img alt="Figure 1" src="/files/ftp_upload/4441/4441fig1.jpg" /> 
Figure 1. Sonicated human genomic DNA fragments in 1% agarose gel. 10 &mu;g of genomic DNA isolated from human iPS cells in 120 &mu;l of 1X TE buffer was sonicated using a sonication device (Covaris). After sonication, 2 &mu;l of the sonicated DNA was loaded onto 1% agarose gel using 100 bp of DNA marker to compare the sizes of the sonicated DNA fragments.
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 <td>Final Concentration</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>_ &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 X &beta;-GT Reaction Buffer</td>
 <td>2 &mu;l</td>
 <td>1 X</td>
 </tr>
 <tr>
 <td>Up to 10 &mu;g genomic DNA</td>
 <td>_ &mu;l</td>
 <td>Up to 500 ng/&mu;l</td>
 </tr>
 <tr>
 <td>UDP-6-N3-Glc (3 mM)</td>
 <td>0.67 &mu;l</td>
 <td>100 &mu;M</td>
 </tr>
 <tr>
 <td>&beta;-GT (40 &mu;M)</td>
 <td>1 &mu;l</td>
 <td>2 &mu;M</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>20 &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
i) For tissue genomic DNA (high 5-hmC content &gt; 0.1%)
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 <td>Final Concentration</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>_ &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 X &beta;-GT Reaction Buffer</td>
 <td>10 &mu;l</td>
 <td>1 X</td>
 </tr>
 <tr>
 <td>Up to 20 &mu;g genomic DNA</td>
 <td>_ &mu;l</td>
 <td>Up to 500 ng/&mu;l</td>
 </tr>
 <tr>
 <td>UDP-6-N3-Glc (3 mM)</td>
 <td>1.33 &mu;l</td>
 <td>100 &mu;M</td>
 </tr>
 <tr>
 <td>&beta;-GT (40 &mu;M)</td>
 <td>2 &mu;l</td>
 <td>2 &mu;M</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>40 &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
ii) For stem cell genomic DNA (median 5-hmC content ~0.05%)
<table border="1">
 <tbody>
 <tr>
 <td>Component</td>
 <td>Volume</td>
 <td>Final Concentration</td>
 </tr>
 <tr>
 <td>Water</td>
 <td>_ &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>10 X &beta;-GT Reaction Buffer</td>
 <td>10 &mu;l</td>
 <td>1 X</td>
 </tr>
 <tr>
 <td>Up to 50 &mu;g genomic DNA</td>
 <td>_ &mu;l</td>
 <td>Up to 500 ng/&mu;l</td>
 </tr>
 <tr>
 <td>UDP-6-N3-Glc (3 mM)</td>
 <td>3.33 &mu;l</td>
 <td>100 &mu;M</td>
 </tr>
 <tr>
 <td>&beta;-GT (40 &mu;M)</td>
 <td>5 &mu;l</td>
 <td>2 &mu;M</td>
 </tr>
 <tr>
 <td>Total volume</td>
 <td>100 &mu;l</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
iii) For cancer cell genomic DNA (low 5-hmC content ~0.01%)
Table 1. Examples of amounts of input DNA and labeling reactions using the samples with various 5-hmC levels by the selective chemical labeling method.
<table border="1">
 <tbody>
 <tr>
 <td>Sample</td>
 <td>5-hmC level</td>
 <td>Starting DNA (&mu;g)</td>
 <td>Recovery after labeling (input to beads) (&mu;g)</td>
 <td>Recovery yield</td>
 <td>Pull-down DNA (ng)</td>
 <td>Pull-down yield</td>
 </tr>
 <tr>
 <td>Adult mouse cerebellum</td>
 <td>0.4%</td>
 <td>10</td>
 <td>7.5</td>
 <td>75%</td>
 <td>236</td>
 <td>3.1%</td>
 </tr>
 <tr>
 <td>Postnatal day 7 mouse cerebellum</td>
 <td>0.1%</td>
 <td>11</td>
 <td>9</td>
 <td>82%</td>
 <td>140</td>
 <td>1.6%</td>
 </tr>
 <tr>
 <td>Mouse ES cell E14</td>
 <td>0.05%</td>
 <td>60</td>
 <td>42</td>
 <td>70%</td>
 <td>350</td>
 <td>0.8%</td>
 </tr>
 </tbody>
</table>
Table 2. Representative results from mouse brain tissues and ES cells.

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No conflicts of interest declared.

5-hydroxymethylcytosine (5-hmC) is a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. The method presented here is for determining the genome-wide distribution of 5-hmC. We use T4 bacteriophage &beta;-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment, and sequencing of 5-hmC-containing DNA fragments in ma...

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>5M Sodium chloride (NaCl)</td>
 <td>Promega</td>
 <td>V4221</td>
 <td></td>
 </tr>
 <tr>
 <td>0.5M pH8.0 Ethylenediaminetetraacetic acid (EDTA)</td>
 <td>Promega</td>
 <td>V4231</td>
 <td></td>
 </tr>
 <tr>
 <td>1M Trizma base (Tris) pH7.5</td>
 <td>Invitrogen</td>
 <td>15567-027)</td>
 <td></td>
 </tr>
 <tr>
 <td>HEPES 1M, pH7.4</td>
 <td>Invitrogen</td>
 <td>15630</td>
 <td></td>
 </tr>
 <tr>
 <td>Magnesium chloride (MgCl2) 1M</td>
 <td>Ambion</td>
 <td>AM9530G</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl sulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>D8418</td>
 <td></td>
 </tr>
 <tr>
 <td>Tween 20</td>
 <td>Fisher BioReagents</td>
 <td>BP337-100</td>
 <td></td>
 </tr>
 <tr>
 <td>DBCO-S-S-PEG3-Biotin conjugate</td>
 <td>Click Chemistry Tools</td>
 <td>A112P3</td>
 <td></td>
 </tr>
 <tr>
 <td>1,4-Dithiothreitol, ultrapure (DTT) Superpure</td>
 <td>Invitrogen</td>
 <td>15508-013</td>
 <td></td>
 </tr>
 <tr>
 <td>QIAquick Nucleotide Removal Kit</td>
 <td>Qiagen</td>
 <td>28304</td>
 <td></td>
 </tr>
 <tr>
 <td>Micro Bio-Spin 6 Column</td>
 <td>Bio-Rad</td>
 <td>732-6222</td>
 <td></td>
 </tr>
 <tr>
 <td>Dynabeads MyOne </td>
 <td>Invitrogen</td>
 <td>650-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Streptavidin C1</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Qiagen MinElute PCR Purification Kit</td>
 <td>Qiagen</td>
 <td>28004</td>
 <td></td>
 </tr>
 <tr>
 <td>UltraPure Agarose</td>
 <td>Invitrogen</td>
 <td>16500500</td>
 <td></td>
 </tr>
 <tr>
 <td>UDP-6-N3-glucose</td>
 <td>Active Motif</td>
 <td>55013</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Enzyme</td>
 </tr>
 <tr>
 <td>&beta;-glucosyltransferase (&beta;-GT)</td>
 <td>New England Biolab</td>
 <td>M0357</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Sonication device</td>
 <td>Covaris</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Desktop centrifuge</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Water bath</td>
 <td>Fisher Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Gel running apparatus</td>
 <td>Bio-Rad</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>NanoDrop1000</td>
 <td>Thermo Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Labquake Tube Shaker</td>
 <td>Barnstead</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Labquake Tube Shaker</td>
 <td>Thermolyne</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Magnetic Separation Stand</td>
 <td>Promega</td>
 <td>Z5342</td>
 <td></td>
 </tr>
 <tr>
 <td>Qubit 2.0 Fluorometer</td>
 <td>Invitrogen</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent setup 
10 X &beta;-GT Reaction Buffer (500 mM HEPES pH 7.9, 250 mM MgCl2) 2 X Binding and washing (B&amp;W) buffer (10 mM Tris pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween 20).</td>
 </tr>
 </tbody>
</table>

selective capture of 5-hydroxymethylcytosine from genomic dna

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Watch this Scientific Journal Video about Selective Capture of 5-hydroxymethylcytosine from Genomic DNA at JoVE.com

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

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11.9K Views.  Emory University School of Medicine. Described is a two-step labeling process using β-glucosyltransferase (β-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.