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.
