About Methylated-DNA IP Kit
The Methylated-DNA IP Kit utilizes immunoprecipitation technology for the enrichment of 5-methylcytosine-containing DNA from any pool of fragmented genomic DNA for use in genome-wide methylation analysis. The kit features a highly specific Anti-5-Methylcytosine Monoclonal Antibody for the capture and separation of methylated DNA from non-methylated DNA in only a few hours (see figure below). Typically, over a hundred-fold enrichment of methylated DNA vs. non-methylated DNA can be achieved with the use of this kit. Recovered DNA is suitable for many downstream applications to analyze genome-wide DNA methylation including PCR, bisulfite treatment, whole-genome amplification, ultra-deep sequencing, and microarray. The product is provided with control DNA and primers for use as a control.
|Processing Time||3 hrs|
|Applications||Utilizes IP technology for the enrichment of 5-methylcytosine-containing DNA with a highly specific anti-5-methylcytosine monoclonal antibody for the "capture" and separation of methylated DNA from non-methylated DNA in only a few hours, from any pool of fragmented genomic DNA for use in genome-wide methylation analysis.|
|Components||Comes as a two component kit: the kit box along with a separate icebox containing the cold-storage components.|
|Enrichment Factor||> 100 fold for Methylated versus Non-methylated DNA.|
|Optimal DNA Input||50 - 500 ng|
Dilute and denature input DNA samples as follows:
Dilute 1-40 µl of sample containing 160 ng of DNA in the DNA Denaturing Buffer to a final volume of 50 µl.
Example: For 32 µl genomic DNA, add 17 µl DNA Denaturing Buffer and 1 µl Control DNA (optional).
Denature the diluted input DNA at 98˚C for 5 minutes.
Complete this step while the DNA is being denatured, or set up tubes before Step 1:
In order, add the following reagents to a 1.5 ml microcentrifuge tube:
a. Add 250 µl MIP Buffer
b. Add 15 µl of ZymoMag Protein A (Pipet up-and-down to expel beads from pipette tip)
Note: ZymoMag Protein A must be resuspended completely by gently flicking and inverting the tube prior to use
c. Add 1.6 µl Mouse Anti-5-Methylcytosine
Invert the tube 2-4 times to mix the antibody/Protein A mixture.
Add the denatured DNA immediately to the antibody/Protein A mixture after Step 1 above is complete.
Incubate the antibody/Protein A/DNA mixture at 37˚C for 0.5-1 hour on a rotator or rocker. Alternatively, invert tubes every 10-15 minutes during the incubation.
Place tubes on a magnetic tube rack, allow time for the beads to cluster, then remove and discard the supernatant.
Add 500 µl of MIP Buffer to each 1.5 ml microcentrifuge tube and secure all the caps. Invert tubes several times and vortex briefly to resuspend the beads. Remove and discard supernatant using the magnetic tube rack.
Repeat wash step (Step 6.) twice more – first with 500 µl MIP Buffer and then with 500 µl of DNA Elution Buffer.
Once the supernatant from the final wash step has been removed and discarded, add 15 µl of DNA Elution Buffer to each tube and resuspend the beads by gently flicking the tube or pipetting up and down. Transfer each bead suspension to a clean 0.2 ml PCR tube.
Incubate the PCR tubes at 75˚C for 5 minutes and follow with a 2-minute spin in a mini-centrifuge.
Transfer the supernatant to new 1.5 ml microcentrifuge tubes without disturbing the beads (if beads are disturbed PCR tubes can be re-spun). This is the recovered DNA.
The recovered DNA is mostly single stranded and suitable for PCR based amplification and other downstream DNA methylation analyses. It can be stored at or below -20°C for later use. For long term storage, it is recommended the DNA be stored at or below -70°C.
The authors utilized the DNA Methylation IP Kit (MeDIP) from Zymo Research to compare DNA methylation results using either MeDIP-Seq or Infinium HumanMethylation450 BeadChIP array. They found the MeDIP-Seq resulted in detection of 15,709 differentially methylation regions, almost twice as many compared the array based method (8070), and concluded that MeDIP-Seq provided a more comprehensive picture of the methylation in human samples.
Researchers used the Methylated DNA IP Kit from Zymo Research to immunoprecipitate methylated DNA from leaf tissue and study the DNA methylation profile in the maize genome. The authors found that specific families of retrotransposons could spread heterochromatin to neighboring genes and epigenetically suppress their expression.
The Methylated-DNA IP Kit was used for CpG immunoprecipitation of genomic DNA isolated from T cells that was sheared to ~200 bp using dsDNA Shearase™ Plus (E2018) to study the DNA methylation status of the Notch-1 promoter.
Enhanced PP2A expression is associated with reduced DNMT1 expression in Systemic Lupus Erythematosus (SLE) patients, so the authors wanted to determine if PP2A may contribute to the DNA hypomethylation observed in SLE patients. Using the Methylated-DNA IP Kit from Zymo Research, the authors were able to enrich for methylated DNA from genomic DNA isolated from T-cells treated with a PP2A suppressor. The enriched DNA was used for real-time PCR to amplify and determine the methylation status of specific genes. The authors found that genes associated with the pathogenesis of SLE were hypomethylated and concluded that enhanced PP2A expression is associated with hypomethylation in SLE patients.
Methylated DNA was enriched from paired-end libraries generated by the authors using the Methylated-DNA IP Kit from Zymo Research and then sequenced using the Illumina HiSeq. The results showed that MeDIP-Seq detected twice as many differentially methylated regions than the Illumina 450k array method.