Name: Methylated DNA Immunoprecipitation (Video) | JoVE Journal
Uploaded: 2009-01-02T00:00:00.000+00:00
Duration: 21 min 24 s
Description: Learn methylated DNA immunoprecipitation (MeDIP) for epigenetics research using anti-5mC antibodies. This two-day protocol isolates methylated DNA fragments to study gene regulation and disease mechanisms.

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5 minutes

We wish to thank members of the Brown and Lam labs for their participation in critiquing this video and article. This work was supported by funds from the Canadian Institutes for Health Research and the Michael Smith Foundation for Health Research.

DNA EXTRACTION AND SAMPLE PREPARATION
DNA from a variety of different samples (cultured cells, fresh frozen as well as formalin-fixed paraffin embedded tissues) can be used for MeDIP. It is important to use highly purified DNA without associated proteins such as histones. It is also important to remove as much RNA as possible from the sample, as it can interfere with both DNA quantitation and antibody binding. The quantity of DNA used for MeDIP can range from 200 ng to 1 &micro;g depending on the amount of DNA available. To demonstrate this protocol, 1 &micro;g of DNA will be used. The following protocol will provide high quality double stranded DNA from cultured cells. Other protocols should be employed for extraction of DNA from other sample types.
<ol>
<li>Add 400 &mu;l of digestion buffer to a cell pellet in a 1.7 ml Eppendorf tube.</li>
<li>Add 100 &mu;g of proteinase K to the tube, and incubate overnight at 50&deg;C.</li>
<li>Add 500 &mu;l phenol pH 7 and mix gently but thoroughly by inverting.</li>
<li>Spin at 13 000 g for 10 mins at room temperature.</li>
<li>Remove aqueous (top) fraction to a new tube.</li>
<li>Repeat Steps 3 through 5 once.</li>
<li>Add 500 &micro;l 1:1 phenol/chloroform pH 7 and mix gently but thoroughly by inverting.</li>
<li>Spin at 13 000 g for 10 mins at room temperature.</li>
<li>Remove aqueous (top) fraction to a new tube.</li>
<li>Add 40 &mu;g of RNase A and incubate 1 hr at 37&deg;C.</li>
<li>Add 500 &mu;l phenol pH 7 and mix gently but thoroughly by inverting.</li>
<li>Spin at 13 000 g for 10 mins at room temperature.</li>
<li>Remove aqueous (top) fraction to a new tube.</li>
<li>Repeat Steps 11 through 13 once.</li>
<li>Add 500 &micro;l 1:1 phenol/chloroform pH 7 and mix gently but thoroughly by inverting.</li>
<li>Spin at 13 000 g for 10 mins at room temperature.</li>
<li>Remove aqueous (top) fraction to a new tube.</li>
<li>Add 1/10th volume 3 M sodium acetate (40 &mu;l) and mix well.</li>
<li>Add 2 volumes (900 &mu;l) of 100% ethanol, mix well, and place at -20&deg;C for 20 mins.</li>
<li>Spin at 13 000 g for 20 mins at 4&deg;C.</li>
<li>Remove ethanol, pulse spin, and remove residual ethanol.</li>
<li>Add 500 &mu;l cold 70% ethanol to wash. Spin at 13 000 g for 20 mins at 4&deg;C.</li>
<li>Remove 70% ethanol, pulse spin, and remove residual ethanol by pipetting.</li>
<li>Air dry the pellet with the cap open for 10 mins at room temperature to remove all traces of residual ethanol.</li>
<li>Resuspend DNA in 50 &mu;l of sterilized dH2O overnight at 4&deg;C.</li>
<li>Quantify the DNA using a NanoDrop Spectrophotometer. An A260:A280 ratio of 1.8 is ideal.</li>
<li>Determine DNA quality and size range on an agarose gel with 100 bp ladder. As little as 10 ng of genomic DNA can be run on a 1.7% agarose gel followed by staining using a dye that is highly sensitive to small amounts of DNA such as SYBR Gold.</li>
<li>In one siliconized tube per sample, prepare 1 &micro;g of DNA in a total volume of 50 &mu;l with the remainder of the volume made up with sterilized dH2O.</li>
</ol>
DNA SONICATION
DNA sonication and the MeDIP protocol must be performed in siliconzied tubes to prevent non specific binding of proteins to tube walls. Optimal sonication times for the DNA samples are based on the degree of DNA sample fragmentation as determined from gel electrophoresis (Step 27). For example, DNA extracted from cultured cells should be of very high molecular weight, and will subsequently require more sonication than DNA extracted from archival samples which are often partially degraded. If samples are of uniformly high molecular weight, it is reasonable at this point to proceed with the sonication as described in this protocol without checking each sample individually. If samples are fragmented as with archival samples, it will be necessary to adapt the sonication procedure likely by decreasing sonication times to obtain 300-100bp fragments. If you expect to process samples that are partially degraded, optimization of sonication parameters can be performed on a representative sample from those of interest. Based on the degree of DNA fragmentation observed from gel electrophoresis (Step 27), the experimenter can predetermine the optimal sonication time for the sample. Here we describe a method to obtain 300-1000 bp DNA fragments by sonication with an automated sonicating device (Bioruptor from Diagenode, UCD-200 TM) using high molecular weight DNA.
<ol>
<li>Water in Biorupter must be at 4&deg;C.</li>
<li>Sonicate for 7 mins on automatic settings (30 sec on 30 sec off at maximum power).</li>
<li>Remove 800 ng (40 &mu;l) of sonicated product and place in siliconized 1.7 ml centrifuge tube for the immunoprecipitation (IP) reaction.</li>
<li>Set aside remaining 200 ng (10 &mu;l) to serve as input (IN) reference DNA (store at 4&deg;C).</li>
</ol>
IMMUNOPRECIPITATON OF METHYLATED DNA
<ol>
<li>Denature the DNA that will be used for IP reaction (800 ng) at 95&deg;C for 10 mins in a water bath.</li>
<li>Cool immediately on ice. Let DNA cool completely (approximately 5 mins on ice) before proceeding with next step.</li>
<li>Add 5 &mu;g monoclonal antibody.</li>
<li>Add IP buffer (used at room temperature throughout this protocol) to a final volume of 500 &mu;l.</li>
<li>Incubate for 2 hrs at 4&deg;C in rotating tube holder.</li>
<li>Just before step 5 is complete, prepare Dynabeads by washing (Steps 6-11). First, resuspend the beads thoroughly in the vial by vortexing.</li>
<li>Transfer 30 &mu;l (~ 2 x 107) of resuspended Dynabeads per reaction plus 1, into a new siliconized tube (for example, if doing 8 reactions, remove enough beads for 9, i.e. 270 &mu;l).</li>
<li>Place the tube on the magnetic rack for 2 mins at room temperature.</li>
<li>Pipette off the supernatant. When removing supernatant, avoid touching the beads against inside wall (where the beads attract to the magnet) with the pipette tip.</li>
<li>Remove the tube from the magnet, and resuspend the beads in an excess volume of IP buffer (750 &mu;l-1000 &mu;l). Place the tube back on the magnetic rack for 2 mins at room temperature.</li>
<li>Repeat the wash once more, and then resuspend the washed beads in IP buffer in the original volume removed in Step 7.</li>
<li>Add 30 &mu;l of washed Dynabeads to each IP reaction</li>
<li>Incubate in a rotating tube holder for 2 hrs at 4&deg;C.</li>
<li>After incubation is complete, place the tube on the magnetic rack for 2 mins at room temperature.</li>
<li>Pipette off the supernatant. Avoid touching the inside wall of the tube (where the beads attract to the magnet) with the pipette tip. Add 500 &mu;l of IP buffer. Mix the tube contents and put it back on the magnetic rack for 2 mins. Repeat wash with 500 ul IP buffer one time.</li>
<li>After removing the supernatant from the last wash, resuspend the beads in 400 &mu;l of digestion buffer.</li>
<li>Treat the reaction with 100 &mu;g of Proteinase K and incubate overnight at 50&deg;C.</li>
</ol>
PURIFICATION OF IMMUNOPRECIPITATED DNA
<ol>
<li>Add 500 &mu;l 1:1 phenol/chloroform pH 7 and vortex thoroughly.</li>
<li>Spin at 13 000 g for 10 mins at room temperature.</li>
<li>Remove aqueous (top) fraction to a new tube.</li>
<li>Repeat Steps 1 through 3 if the interphase between the aqueous and organic layers appears cloudy.</li>
<li>Add 1/10th volume 3 M sodium acetate (40 &mu;l) and vortex.</li>
<li>Add 1 &mu;l of glycogen (20 &mu;g/&mu;l) and vortex.</li>
<li>Add 2 volumes (1000 &mu;l) of 100% ethanol, vortex, and place at -20&deg;C for 20 mins.</li>
<li>Spin at 13 000 g for 20 mins at 4&deg;C.</li>
<li>Remove ethanol, pulse spin, and remove residual ethanol.</li>
<li>Add 500 &mu;l cold 70% ethanol to wash. Vortex briefly, and spin at 13 000 g for 20 mins at 4&deg;C.</li>
<li>Remove 70% ethanol, pulse spin, and remove residual ethanol by pipetting.</li>
<li>Air dry the pellet with the cap open for 10 mins at room temperature to remove all traces of residual ethanol.</li>
<li>Resuspend DNA pellet in 10 &mu;l sterilized dH20. </li>
</ol>
VALIDATION BY PCR
<ol>
<li>You may test to ensure that your MeDIP procedure is working by performing the MeDIP protocol using normal human DNA (male or female), and subsequently assaying a region known to be enriched for methylation.</li>
<li>Remove 30% of MeDIP product to PCR tube, and another 30% of MeDIP product to another PCR tube.</li>
<li>Put 10 ng of IN DNA into a PCR tube, and 10 ng of IN DNA into another PCR tube. You should now have four separate PCR reactions to set up.</li>
<li>Perform PCR using H19 and CTRL primers as indicated in Table 1.</li>
<li>Prepare two mastermixes (one for each primer set) for PCR reactions with 12.5 &micro;l total volume as outlined in Table 2.</li>
<li>Thermocycle the PCR using the conditions outlined in Table 3.</li>
<li>Run 5 &micro;l of PCR products on a 2% agarose gel for visualization.</li>
<li>The expected results for successful MeDIP are shown in Table 4.</li>
</ol>
Tables
Table 1: H19 and CTRL primers for PCR validation. 
<table border="1">
<tbody>
<tr>
<td>Primer Set</td>
<td>Forward Primer</td>
<td>Reverse Primer</td>
<td>Anticipated Product Size</td>
</tr>
<tr>
<td>H19</td>
<td>H19_F 5&rsquo;-cgagtgtgcgtgagtgtgag</td>
<td>H19_R 5&rsquo;-ggcgtaatggaatgcttgaa</td>
<td>174 bp</td>
</tr>
<tr>
<td>CTRL (control)</td>
<td>CTRL_F5&rsquo;-gagagcattagggcagacaaa</td>
<td>CTRL_R 5&rsquo;-gttcctcagacagccacattt</td>
<td>139 bp</td>
</tr>
</tbody>
</table>

Table 2. Mastermixes for PCR reactions. 
<table border="1">
<tbody>
<tr>
<td>&nbsp;</td>
<td>H19 Mix (per Rxn)</td>
<td>CTRL Mix (per Rxn)</td>
</tr>
<tr>
<td>ddH2O</td>
<td>6.875</td>
<td>6.875</td>
</tr>
<tr>
<td>10X Buffer</td>
<td>1.25</td>
<td>1.25</td>
</tr>
<tr>
<td>dNTP mix (10 mM each)</td>
<td>0.25</td>
<td>0.25</td>
</tr>
<tr>
<td>MgCl2 (50 mM)</td>
<td>0.25</td>
<td>0.25</td>
</tr>
<tr>
<td>Primers (H19_F/R or CTRL_F/R) (10 &micro;M each F/R)</td>
<td>0.625</td>
<td>0.625</td>
</tr>
<tr>
<td>Platinum Taq</td>
<td>0.25</td>
<td>0.25</td>
</tr>
</tbody>
</table>

Table 3. PCR thermocyling conditions. 
<table border="1">
<tbody>
<tr>
<td>&nbsp;</td>
<td>95&deg;C</td>
<td>5:00</td>
</tr>
<tr>
<td rowspan="3">40X </td>
<td>95&deg;C</td>
<td>0:30</td>
</tr>
<tr>
<td>56&deg;C</td>
<td>0:30</td>
</tr>
<tr>
<td>72&deg;C</td>
<td>0:15</td>
</tr>
</tbody>
</table>

Table 4. Expected PCR results for successful MeDIP. 
<table border="1">
<tbody>
<tr>
<td>&nbsp;</td>
<td colspan="2" align="center">Template DNA</td>
</tr>
<tr>
<td>Primer used</td>
<td>IN</td>
<td>IP</td>
</tr>
<tr>
<td>H19</td>
<td>Positive</td>
<td>Positive</td>
</tr>
<tr>
<td>CTRL</td>
<td>Positive</td>
<td>Negative</td>
</tr>
</tbody>
</table>

<ol><li>Beck, S., Rakyan, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+methylome%3A+approaches+for+global+DNA+methylation+profiling%5BTitle%5D)+AND+%22Trends+Genet%22%5BJournal%5D)">The methylome: approaches for global DNA methylation profiling.</a> Trends Genet. 24, 231-237 (2008).</li>
<li>Lu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetics%2C+disease%2C+and+therapeutic+interventions%5BTitle%5D)+AND+%22Ageing+research+reviews%22%5BJournal%5D)">Epigenetics, disease, and therapeutic interventions.</a> Ageing research reviews. 5, 449-467 (2006).</li>
<li>Zilberman, D., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genome-wide+analysis+of+DNA+methylation+patterns%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Genome-wide analysis of DNA methylation patterns.</a> Development. 134, 3959-3965 (2007).</li>
<li>Feinberg, A. P., Tycko, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+history+of+cancer+epigenetics%5BTitle%5D)+AND+%22Nature+reviews%22%5BJournal%5D)">The history of cancer epigenetics.</a> Nature reviews. 4, 143-153 (2004).</li>
<li>Weber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distribution%2C+silencing+potential+and+evolutionary+impact+of+promoter+DNA+methylation+in+the+human+genome%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome.</a> Nat Genet. 39, 457-466 (2007).</li>
<li>Weber, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosome-wide+and+promoter-specific+analyses+identify+sites+of+differential+DNA+methylation+in+normal+and+transformed+human+cells%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells.</a> Nat Genet. 37, 853-862 (2005).</li>
<li>Wilson, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenomics%3A+mapping+the+methylome%5BTitle%5D)+AND+%22Cell+Cycle%22%5BJournal%5D)">Epigenomics: mapping the methylome.</a> Cell Cycle. 5, 155-158 (2006).</li>
<li>Gazin, C., Wajapeyee, N., Gobeil, S., Virbasius, C. M., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+elaborate+pathway+required+for+Ras-mediated+epigenetic+silencing%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">An elaborate pathway required for Ras-mediated epigenetic silencing.</a> Nature. 449, 1073-1077 (2007).</li>
<li>Karpinski, P., Sasiadek, M. M., Blin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aberrant+epigenetic+patterns+in+the+etiology+of+gastrointestinal+cancers%5BTitle%5D)+AND+%22Journal+of+applied%22%5BJournal%5D)">Aberrant epigenetic patterns in the etiology of gastrointestinal cancers.</a> Journal of applied. 49, 1-10 (2008).</li>
<li>Maekawa, M., Watanabe, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetics%3A+relations+to+disease+and+laboratory+findings%5BTitle%5D)+AND+%22Current+medicinal+chemistry%22%5BJournal%5D)">Epigenetics: relations to disease and laboratory findings.</a> Current medicinal chemistry. 14, 2642-2653 (2007).</li>
<li>Vucic, E. A., Brown, C. J., Lam, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetics+of+cancer+progression%5BTitle%5D)+AND+%22Pharmacogenomics%22%5BJournal%5D)">Epigenetics of cancer progression.</a> Pharmacogenomics. 9, 215-234 (2008).</li>
<li>Egger, G., Liang, G., Aparicio, A., Jones, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epigenetics+in+human+disease+and+prospects+for+epigenetic+therapy%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Epigenetics in human disease and prospects for epigenetic therapy.</a> Nature. 429, 457-463 (2004).</li>
<li>Jones, P. A., Baylin, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+fundamental+role+of+epigenetic+events+in+cancer%5BTitle%5D)+AND+%22Nat+Rev+Genet%22%5BJournal%5D)">The fundamental role of epigenetic events in cancer.</a> Nat Rev Genet. 3, 415-428 (2002).</li>
<li>Fraga, M. F., Esteller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DNA+methylation%3A+a+profile+of+methods+and+applications%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">DNA methylation: a profile of methods and applications.</a> Biotechniques. 33, 632-649 (2002).</li>
</ol>

There is a growing awareness of the significant role DNA methylation plays in disease, therefore the development of assays to measure this modification are becoming increasingly important 3, 12, 13. The MeDIP technique is an amenable tool for screening at both the whole-genome and locus-specific level 6, 7. This technique provides a rapid view of DNA methylation levels using limited amounts of starting DNA and allows for easy comparisons between different sources. Downstream applications using th...

<table><tbody><tr><td>Biorupter sonicator</td><td>Tool</td><td>Diagenode</td><td>UCD-200 TM</td><td></td></tr><tr><td>1.7ml SafeSeal Microcentrifuge Tubes</td><td>Other</td><td>Sorenson BioScience</td><td>11510</td><td></td></tr><tr><td>ND 3300 Spectrophotometer</td><td>Tool</td><td>NanoDrop</td><td></td><td></td></tr><tr><td>Primary Antibody: Anti-5&amp;rsquo;-methylcytosine mouse mAb</td><td>Reagent</td><td>Calbiochem</td><td>162 33 D3</td><td></td></tr><tr><td>Secondary Antibody: Dynabeads M-280 Sheep anti-mouse IgG</td><td>Reagent</td><td>Invitrogen</td><td>112-01D</td><td></td></tr><tr><td>Magnetic Tube Rack</td><td>Tool</td><td>Invitrogen</td><td>CS15000</td><td></td></tr><tr><td>Mini LabRoller</td><td>Tool</td><td>Labnet International</td><td>H5500</td><td></td></tr><tr><td>IP Buffer</td><td></td><td></td><td></td><td>10 mM NaPO&lt;sub&gt;4&lt;/sub&gt; pH 7.0, 140 mM NaCl, 0.05% Triton X-100. Stored at room temperature</td></tr><tr><td>Digestion Buffer</td><td></td><td></td><td></td><td>10 mM Tris pH8.0, 100 mM EDTA, 0.5% SDS, 50 mM NaCl</td></tr></tbody></table>

methylated dna immunoprecipitation

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

This video demonstrates a protocol for methylated DNA immunoprecipitation or meetup. A method used to specifically isolate methylated DNA in an unbiased manner. DNA methylation in mammals is characterized by the addition of a methyl group to the C five position of cytosine.When present in A CPG dinucleotide, it is a reversible epigenetic mark that plays an important role in normal development, as well as several disease processes. Normal functions of DNA methylation include X chromosome inactivation in females, repetitive element silencing, genomic imprinting, and maintenance of genomic stability. DNA methylation has also been shown to play a role in numerous diseases such as cancer, der Willi syndrome, engelman syndrome, and immunodeficiency, centri meric instability and facial anomaly syndrome.In cancers, for example, abnormal hypermethylation and promoters can result in the silencing of vital tumor suppressor genes. While improper hypomethylation results in chromosomal instability and oncogene activation meetup is accomplished by first sonic skating DNA into fragments ranging in size from 300 to 1000 base pairs. The sonicated DNA is then denatured at 95 degrees Celsius.Primary antibodies, which recognize five prime methazine are added and allowed to bind methylated DNA fragments. Secondary antibodies, conjugated to magnetic dyna beads are used to isolate fragments of DNA, which have been bound by the primary antibody. Unmethylated fragments are removed and the antibodies are digested by proteinase.KA phenyl chloroform cleanup removes the digested protein and precipitation purifies the DNA, which is now enriched for methylated fragments. Meetup, DNA may be used for applications such as PCR microarray and sequencing. Meetup is generally performed as a two day procedure.Day one involves DNA preparation, sonication immunoprecipitation and protein digestion. Day two involves pH chloroform extraction and ethanol precipitation to purify and isolate the DNA enriched for methylation. There are a few special materials that are required for meetup, including siliconized centrifuge tubes, A DNA sunation device, primary antibody, anti five prime methazine mouse, monoclonal antibody, secondary antibody, dyna beads, conjugated to sheep, anti-US IgG, and a magnetic tube rack.The first step in The meetup protocol is the preparation of DNA. It is imperative to assess the nature of the DNA beforehand. Knowing the state of the sample DNA, whether it is primarily double or single stranded is required as accurate quantitation of the sample is of the utmost importance and various methods of determining DNA quantity can be adversely affected by the presence of undesired DNA species.Double stranded DNA quantity can be assessed easily by spectral photometry using a NanoDrop 3, 300 nd load 1.5 microliters and set the measurement type to nucleic acid, select DNA 50 as the calculation and initialize the instrument with 1.5 microliters of water. Clean the pedestals and blank the instrument with 1.5 microliters of the liquid. The sample is dissolved in clean the pedestals load 1.5 microliters of the sample and hit measure.The concentration will be given and can be used. To calculate the number of microliters needed for me, dip an A two 60 to two 80 ratio of 1.8 is ideal here. A one microgram sample is prepared.However, a sample of as little as 200 nanograms is sufficient to obtain successful results. For meetup, Load the DNA sample into a clean siliconized tube. Add enough sterile water To bring the final volume to 50 microliters.DNA Sonication and immunoprecipitation must be performed in siliconized tubes to prevent nonspecific binding of DNA and proteins to tube walls. Once the DNA has been prepared, the sample must be sonic sonication is required to fragment the DNA as immunoprecipitation is more efficient with smaller fragments of DNA, load the sample into the sonicate, ensure that the power setting is set to high, and that the machine is set to 30 seconds. On 30 seconds off, fill the base into the fill line with four degrees Celsius water.Sonicate the sample For seven minutes. When Sonication is finished, remove the tube and put it on ice. It is important to optimize Sonication conditions for your anticipated sample, DNA size beforehand.For example, a partially degraded DNA sample will require a shorter sonication time. Sonication products can be visualized by standard electrophoretic techniques. DNA fragments ranging in size from 300 to 1000 base pairs in length are desired at this point for later use.In microarray and PCR assays, you should reserve one fifth of your DNA to serve as the input or reference DNA in this case, 800 nanograms of sonicated DNA will be used for the immunoprecipitation and the remainder will be set aside as reference DNA. The next step in the media protocol is the immunoprecipitation or IP reaction before the addition of the primary antibody, the DNA must be denatured. Denaturation is required because the primary antibody is sensitive to single stranded DNA.Place the sample in a water bath at 95 degrees Celsius for 10 minutes. When the denaturation step is complete, immediately transfer the tube to ice for at least five minutes. This will prevent the denature DNA from Reen kneeling and will allow for optimal antibody binding When the sample has cooled.Spin the tube briefly to draw the contents to the bottom. Next, the primary antibody should Be added. The amount of antibody Used should be titrated for each batch and for each manufacturer.In this case, we add five micrograms of primary antibody and then add immunoprecipitation buffer or IP buffer to a final volume of 500 microliters. Next, The IP DNA is placed in a rotator and left to incubate at four degrees Celsius for two hours. Rotation is required to prevent antibody conglomeration within the tube during the incubation.About 15 minutes before the primary antibody incubation is complete, clean and prepare the magnetic dyna beads. First, thoroughly resuspend the beads in the vial By vortexing. Then Transfer 30 microliters of beads into a clean siliconized tube and place that tube on a magnetic rack for multiple reactions.Transfer 30 microliters of beads per reaction, plus enough for one additional reaction to allow for pipetting error or inadvertent loss of some beads. For example, for eight reactions. Remove enough beads for nine reactions or 270 microliters.The magnetic beads will be drawn towards the wall of the magnetic rack. Allow two minutes for the beads to migrate and settle into a defined streak on The tube wall. Next, remove And discard the supernatant pipette carefully being sure to avoid disturbing the beads on the back wall with the Pipette tip.Replace the supernatant With an excess volume of IP buffer vortex and put it back on the magnetic rack for two Minutes. Repeat this wash once more and resuspend the wash beads in their original volume of IP buffer. Retrieve the sample tube from the Rotator when the incubation is finished.To complete the immunoprecipitation, add 30 microliters of clean beads to the reaction and place the tube back in a rotator for two hours at four degrees Celsius. During this incubation, the secondary antibody, which is anti mouse IgG, will bind to the primary mouse antibody. The anti mouse antibodies are conjugated to magnetic dyna beads, allowing for easy recovery of the bound products and the purification step to follow.Once the incubation with both antibodies is complete, place the IP tube on the magnetic rack for two minutes. At this point, the methylated DNA will be bound by the primary antibody, which itself will be bound by the dyna bead conjugated secondary antibody. Remove and discard the supernatant and replace it with 500 microliters of IP buffer.Mix the contents of the tube and place it back on the rack for two minutes. Repeat this wash with IP buffer once more. After removing the supernatant from the last wash Resus, suspend the beads for the final time.In 400 microliters of digestion Buffer, treat the reaction with 100 micrograms of proteinase K and incubate overnight at 50 degrees Celsius. The proteinase K treatment is necessary to digest the antibody proteins and any residual protein associated with the DNA, allowing the methylated DNA to be released into solution. DNA purification is carried out on the second day of the meetup procedure, retrieve The digest tube from the incubator.In a fume hood, add 500 microliters of one-to-one pheno chloroform at pH seven to the tube and vortex thoroughly Spin the tube for 10 minutes at 13, 000. G.Carefully Remove the top aqueous phase from the tube without disturbing the interface or removing any of the organic layer. Transfer the aqueous phase to a new tube.A second extraction may be done if the interface Appears cloudy. Next, add one 10th volume of three molar sodium acetate in this case, 40 microliters. One microliter Of glycogen may be used as a carrier to facilitate pellet cleanup.Vortex the tube to Mix the contents. Then add Two volumes of 100%ethanol, vortex and freeze in a minus 20 degrees Celsius freezer for 20 minutes After freezing. The precipitated DNA is spun down in a centrifuge at 13, 000 G and four degrees Celsius for 20 minutes.A DNA pellet will form at the bottom of the tube. Retrieve the tube from the Centrifuge and remove the ethanol. Be careful not to disturb the pellet at the bottom of the tube.A pulse spin may be necessary to remove any residual Ethanol. To wash the pellet, Add 500 microliters of cold, 70%ethanol vortex, and spin again for 20 minutes at 13, 000 G and four degrees Celsius. When the second spin Is complete, remove the 70%ethanol pulse spin and remove the residual ethanol with A pipette.Leave the Tube on a rack with the cap open to dry the pellet and to allow the remaining ethanol to Evaporate. Finally, Reus suspend the pellet containing only DNA enriched for methylation and 10 microliters of sterile water, allowing sufficient time for the DNA pellet to dissolve before use. It is good practice to Validate the meetup proficiency by PCR.This can be done by running PCR reactions designed to amplify products that illustrate methylated and unmethylated DNA fragments differentially for normal human DNA primers amplifying. The H 19 imprinting control region may be used in conjunction with primers designed to amplify a region of the genome, which is not methylated. Example, primer sequences and PCR cycling conditions may be found in the accompanying protocol.At this point, it is necessary to retrieve the input or reference DNA that was set aside at the beginning of the meetup protocol. One set of PCR reactions will be set up using the input DNA, and the other set will be using the IP DNA enriched for methylation. If the meetup was successful, the IP PCR will produce a product using the methylation primers and no product using the control primers.Conversely, the input PCR should produce products using both primers as it contains both methylated and unmethylated. DNA fragments. DNA enriched for methylated fragments can be used for a number of downstream applications, such as PCR sequencing and microarray analysis.When integrated with other molecular biology techniques, it can be used to investigate the methylation status of DNA in a locus specific or genome wide manner for methylation profiling or studying Epigenomics.

Research

JoVE Journal

Biology

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNA Methylation: Bisulphite Modification and Analysis

This video describes the technique of dot-blot to determine the level of DNA methylation in human chondrocytes in vitro via the blotting of DNA samples on a nylon membrane and the subsequent visualization using monoclonal antibodies. This helps to determine the chondrocytes' phenotype at various stages in culture.

5-Methylcytosine Dot Blot to Determine the Extent of DNA Methylation

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

Methyl-binding DNA capture Sequencing for Patient Tissues

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Chromatin immunoprecipitation (ChIP) is a powerful tool for understanding the molecular mechanisms of gene regulation. However, the method involves difficulties in obtaining reproducible chromatin fragmentation by mechanical shearing. Here, we provide an improved protocol for a ChIP assay using enzymatic digestion.

Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Learn methylated DNA immunoprecipitation (MeDIP) for epigenetics research using anti-5mC antibodies. This two-day protocol isolates methylated DNA fragments to study gene regulation and disease mechanisms.

Methylated DNA Immunoprecipitation

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