Methylated DNA Immunoprecipitation - Methods - Methylated DNA Immunoprecipitation (MeDIP)

Methylated DNA Immunoprecipitation (MeDIP)

Genomic DNA is extracted (DNA extraction) from the cells and purified. The purified DNA is then subjected to sonication to shear it into random fragments. This sonication process is quick, simple, and avoids restriction enzyme biases. The resulting fragments range from 300 to 1000 base pairs (bp) in length, although they are typically between 400 and 600 bp. The short length of these fragments are important in obtaining adequate resolution, improving the efficiency of the downstream step in immunoprecipitation, and reducing fragment-length effects or biases. Also, the size of the fragment affects the binding of 5-methyl-cytidine (5mC) antibody because the antibody needs more than just a single 5mC for efficient binding. To further improve binding affinity of the antibodies, the DNA fragments are denatured to produce single-stranded DNA. Following denaturation, the DNA is incubated with monoclonal 5mC antibodies. The classical immunoprecipitation technique is then applied: magnetic beads conjugated to anti-mouse-IgG are used to bind the anti-5mC antibodies, and unbound DNA is removed in the supernatant. To purify the DNA, proteinase K is added to digest the antibodies and release the DNA, which can be collected and prepared for DNA detection.

For more details regarding the experimental steps see.