1Department of Cancer Genetics and Developmental Biology,BC Cancer Research Centre, 2Interdisciplinary Oncology Program,University of British Columbia – UBC, 3Department of Pathology and Laboratory Medicine,University of British Columbia – UBC, 4Photography/Video Production, Multi-Media Services,BC Cancer Agency, 5Department of Medical Genetics, Life Sciences Institute,,University of British Columbia – UBC
Summary
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).
Abstract
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.
Protocol
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 µg depending on…
Discussion
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…
Acknowledgements
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.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Biorupter sonicator | Tool | Diagenode | UCD-200 TM | |
| 1.7ml SafeSeal Microcentrifuge Tubes | Other | Sorenson BioScience | 11510 | |
| ND 3300 Spectrophotometer | Tool | NanoDrop | ||
| Primary Antibody: Anti-5′-methylcytosine mouse mAb | Reagent | CalBiochem | 162 33 D3 | |
| Secondary Antibody: Dynabeads M-280 Sheep anti-mouse IgG | Reagent | Invitrogen | 112-01D | |
| Magnetic Tube Rack | Tool | Invitrogen | CS15000 | |
| Mini LabRoller | Tool | Labnet International | H5500 | |
| IP Buffer | 10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100. Stored at room temperature | |||
| Digestion Buffer | 10 mM Tris pH8.0, 100 mM EDTA, 0.5% SDS, 50 mM NaCl |
References
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- Weber, M., et al. Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet. 37, 853-862 (2005).
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Tags
Methylated DNA ImmunoprecipitationDNA Methylation PatternsEpigeneticsGene ExpressionNormal DevelopmentDiseaseMethylation ProfilesMeDIPMethylated DNA ExtractionAntibody ReactionImmunoprecipitated DNAInput DNAHeat DenaturationAnti-5’mC AntibodyMagnetic BeadsSecondary AntibodyAntibody ComplexSeparation Of Methylated DNANon-methylated DNA Removal
Cite This Article
Thu, K. L., Vucic, E. A., Kennett, J. Y., Heryet, C., Brown, C. J., Lam, W. L., Wilson, I. M. Methylated DNA Immunoprecipitation. J. Vis. Exp. (23), e935, doi:10.3791/935 (2009).