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).
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 the amount of DNA available. To demonstrate this protocol, 1 µg 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.
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.
IMMUNOPRECIPITATON OF METHYLATED DNA
PURIFICATION OF IMMUNOPRECIPITATED DNA
VALIDATION BY PCR
Tables
Table 1: H19 and CTRL primers for PCR validation.
Primer Set | Forward Primer | Reverse Primer | Anticipated Product Size |
H19 | H19_F 5’-cgagtgtgcgtgagtgtgag | H19_R 5’-ggcgtaatggaatgcttgaa | 174 bp |
CTRL (control) | CTRL_F5’-gagagcattagggcagacaaa | CTRL_R 5’-gttcctcagacagccacattt | 139 bp |
Table 2. Mastermixes for PCR reactions.
H19 Mix (per Rxn) | CTRL Mix (per Rxn) | |
ddH2O | 6.875 | 6.875 |
10X Buffer | 1.25 | 1.25 |
dNTP mix (10 mM each) | 0.25 | 0.25 |
MgCl2 (50 mM) | 0.25 | 0.25 |
Primers (H19_F/R or CTRL_F/R) (10 µM each F/R) | 0.625 | 0.625 |
Platinum Taq | 0.25 | 0.25 |
Table 3. PCR thermocyling conditions.
95°C | 5:00 | |
40X |
95°C | 0:30 |
56°C | 0:30 | |
72°C | 0:15 |
Table 4. Expected PCR results for successful MeDIP.
Template DNA | ||
Primer used | IN | IP |
H19 | Positive | Positive |
CTRL | Positive | Negative |
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 the MeDIP product include a variety of microarrays such as whole genome and CpG island oligonucleotide arrays, direct PCR assays for loci of interest, as well as sequencing.
MeDIP provides a distinct approach to DNA methylation detection that relies upon antibodies to distinguish methylated and unmethylated DNA 6. While MeDIP is faster than traditional bisulfite sequencing approaches and it is not limited to the analysis of specific sequences like restriction enzyme analysis, the immunoprecipitation will be dependent on DNA sequence including CpG density, repetitive element presence and composition. Thus, appropriate controls, as with every experiment, are important to the analysis and interpretation of MeDIP results.
There are several steps throughout the MeDIP technique where extra care must be taken. These include: the use of siliconized tubes to prevent non-specific binding of DNA to tube walls; ensuring adequate fragmentation of DNA after sonication; and ensuring that DNA is completely denatured. Additionally, note that quantification of single-stranded MeDIP product may be difficult because there is a limited amount of material and many common techniques for estimating DNA quantity, such as spectrophotometry, work best with double-stranded DNA. Additionally, it is important to observe proper laboratory safety practices at all times, especially when caustic substances such as phenol are used.
The MeDIP technique can also be modified at several steps. Importantly the protocol can be scaled up or down to allow increased yields, or work with limited sample sizes. An alternative fragmentation approach, such as the use of restriction enzymes (e.g. AluI digestion), reduces equipment requirements, but can also introduce biases potentially limiting DNA pull down in some regions. As with any approach, results are best validated using an alternative approach to DNA methylation detection 1, 14.
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.
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 |