The MeDIP Kit is designed to immunoprecipitate and enrich for single-stranded DNA fragments containing 5-methylcytosine (5-mC). The MeDIP Kit contains a monoclonal 5-methylcytidine antibody, a bridging antibody and the necessary buffers to perform methylated DNA immunoprecipitation (MeDIP). The affinity of the antibody enables separation of 5-mC DNA from 5-hmC DNA using single-stranded input DNA. For complete details, click the 5-mC Info tab below.
Active Motif's fast, magnetic protocol has been streamlined to minimize the number of wash and incubation steps, saving you valuable time. The kit also includes MseI digested human genomic DNA and PCR primers that can be used to verify the efficiency of the enrichment. Additionally, the kit contains a bar magnet for easy separation and elution of the enriched methylated DNA. Click the Data tab below to see results from the kit; manuals can be downloaded under the Documents tab.
5-Methylcytosine (5-mC) MeDIP
Methylated DNA Immunoprecipitation (MeDIP) is an immunocapture technique in which an antibody specific for methylated cytosines is used to immunoprecipitate methylated genomic DNA fragments. MeDIP uses single-stranded DNA that has been fragmented by sonication or enzymatic digestion. The addition of a monoclonal antibody specific for 5-methylcytosine in the presence of a bridging antibody enables capture of the DNA fragments containg 5-mC methylation. The use of Protein G magentic beads provides quick and efficient capture. Methylated fragments are then eluted from the magnetic beads providing DNA samples that are specifically enriched for 5-mC DNA methylation. This selectivity is important as most common approaches to analyze DNA methylation, such as enzymatic approaches and bisulfite conversion, are unable to distinguish between 5-mC and 5-hmC.
The affinity of the antibody used in the MeDIP kit enables detection of methylated cytosines regardless of their context. This means that the methylation does not need to occur in the context of a CpG dinucleotide, unlike methyl-binding protein (MBD) affinity enrichment methods which requires CpG methylation for proper detection.
MeDIP Kit Advantages
- Specific antibody only enriches 5-mC methylated DNA
- Includes bridging antibody to increase efficiency of DNA capture
- One-step IP reaction
- No need to pre-wash magnetic beads
- Optimized magnetic protocol to minimize hands-on time
- Kit includes positive control human genomic DNA and PCR primers to verify efficiency
Flow Chart of MeDIP Method
Figure 1: Flow chart of the MeDIP Method.
Single-stranded, fragmented genomic DNA containing 5-methylcytosine can be specifically captured from the rest of the genomic DNA with the mononclonal 5-mC antibody in the presence of a bridging antibody and magnetic beads. Following an overnight incubation, the enriched DNA is then eluted from the beads. (Click image to enlarge.)
The MeDIP Kit is able to selectively enrich for DNA fragments containing 5-methylcytosine DNA methylation from the rest of the genomic DNA population. The MeDIP Kit was tested using 500 ng of the included MseI digested human genomic DNA in the presence or absence of bridging antibody. Eluted DNA was purified with Active Motif's Chromatin IP DNA Purification Kit (Catalog No. 58002) and tested in real time PCR with the included ZC3H13 PCR primer set. Results shown in Figure 1 demonstrate the importance of using a bridging antibody within the assay. In Figure 2, enriched DNA was tested using either the included positive control ZC3H13 PCR primer mix or a negative control GAPDH PCR primer mix.
Figure 1: MeDIP Kit in the presence or absence of bridging antibody.
MseI digested human genomic DNA (500 ng) was processed using the MeDIP Kit with the 5-methylcytidine mAb or negative control mouse IgG in the presence or absence of bridging antibody. Eluted DNA was purified and tested using real time PCR with the included ZC3H13 PCR primer mix. The 5-methylcytidine antibody specifically enriched methylated DNA in the presence of bridging antibody, but did not enrich for DNA with the mouse IgG or in the absence of bridging antibody. The ZC3H13 locus analyzed in this experiment is methylated in human genomic DNA and therefore should amplify. Data shown are the results from samples assayed in duplicate. These results are provided for demonstration only.
Figure 2: Real time PCR results of the control DNA with ZC3H13 and GAPDH PCR primer sets.
MseI digested human genomic DNA (500 ng) was processed using the MeDIP Kit with the 5-methylcytidine mAb or negative control mouse IgG. Eluted DNA was column purified with Active Motif's Chromatin IP DNA Purification Kit (Catalog No. 58002) and tested using real time PCR with the included ZC3H13 PCR primer mix and a negative control GAPDH PCR primer mix. The 5-methylcytidine antibody specifically enriched methylated DNA with the ZC3H13 locus but did not enrich for DNA with either the GAPDH locus or the mouse IgG. Data shown are the results from samples assayed in duplicate. These results are provided for demonstration only.
Preparation of MeDIP Enriched Samples for Next-Gen Sequencing
Since MeDIP uses a 5-methylcytosine antibody that only binds methylated cytosines in the context of single-stranded DNA to enrich for methylated genomic DNA, the DNA must be denatured prior to immunoprecipitation. As a result of denaturation, the enriched DNA can not be processed for Next-Gen sequencing using the typical sequencing library generation protocols, as these require adapter ligation to double-stranded DNA. This problem can be circumvented by addition of the Next-Gen adapters to fragmented genomic DNA prior to performing MeDIP.
If you intend to perform Next-Gen sequencing analysis following methylated DNA immunoprecipitation using the Active Motif MeDIP kit, an optimized library generation protocol adapted from the Illumina Paired-End DNA Sample Prep Kit protocol is provided in the MeDIP manual. Just follow the simple instructions for library preparation, immunoprecipitation, and PCR amplification to prepare your MeDIP enriched DNA for analysis by Next-Gen Sequencing.
Click on image to enlarge size.
Figure 1: Next-Gen sequencing data generated using Active Motif’s MeDIP Kit correlates well with CpG density.
DNA was enriched from 1 ug of adaptor ligated human PBMC DNA using Active Motif’s MeDIP Kit. Next-Gen sequencing was performed using the Illumina platform to generate 26 million sequence tags. Tags were mapped to generate a whole-genome DNA methylation profile. The image above shows that the enriched regions (purple peaks) correlate well with CpG density.
Click on image to enlarge size.
Figure 2: Next-Gen sequencing data generated using Active Motif’s MeDIP Kit detects methylation at CpG shores.
DNA was enriched from 1 ug of adaptor ligated human PBMC DNA using Active Motif’s MeDIP Kit. Next-Gen sequencing was performed using the Illumina platform to generate 26 million sequence tags. Tags were mapped to generate a whole-genome DNA methylation profile. The image above shows two examples of DNA methylation being detected at CpG shores rather than in the CpG island itself. This data agrees with recent findings showing that a large portion of methylated sites occur in these regions that are adjacent to CpG islands.
Contents & Storage
The MeDIP Kit contains a mouse monoclonal 5-methylcytidine antibody, a bridging antibody, negative control mouse IgG, PIC, Buffer C, Buffer D, Elution Buffer AM2, Neutralization Buffer, Sterile water, PCR tubes, Protein G magnetic beads and a bar magnet for the separation and elution of DNA fragments containing 5-mC. Protocols are provided for the fragmentation of sample DNA all the way through to real time PCR analysis of a locus of interest.
The kit also includes positive control DNA and PCR primers. Human genomic DNA, MseI digested and ZC3H13 PCR primer pair are provided for use as a p