DNA immunoprecipitation is a large-scale enrichment technique in molecular biology that is used to isolate DNA fragments that harbor a specific modification. Antibodies raised against a specific modified purine or pyrimidine are used to capture that base from a pool of genomic DNA molecules, and its genomic context can then be investigated.
To perform DNA immunoprecipitation, purified DNA is sonicated, causing it to shear into random fragments with an average size of 500 bp (Jacinto, Ballestar, & Esteller, 2007). Depending on the characteristics of the antibody, sheared DNA may or may not be denatured and is then incubated with antibody against the specific modification of interest. The classic immunoprecipitation technique is then used: magnetic beads are used to pull the primary antibody with bound DNA fragments. After removing unbound DNA from supernatant, proteinase K is used to release DNA from antibody and magnetic beads, and DNA is then collected. Various methods, including sequencing and quantitative PCR, can be used to search for loci rich in the modification of interest.
In 2005, this technology was first described to identify sites of differential DNA methylation in normal and transformed human cells by using an antibody against 5mC (Weber et al., 2005). Thereafter, antibodies were developed that detect other modified DNA bases; for example, antibody against 5hmC, 5fC, 5caC, 6md, and several others (Fu et al., 2015; Inoue, Shen, Dai, He, & Zhang, 2011; Li et al., 2014b; Pfaffeneder et al., 2011; Szwagierczak, Bultmann, Schmidt, Spada, & Leonhardt, 2010). Therefore, one of the main advantages of this technique is that it is not limited to 5mC, 5hmC, or both, nor is it greatly affected by the chemical structure or reactivity of any particular DNA modification. Moreover, it does not require any chemical conversation, thereby eliminating concerns about incomplete treatment and vastly reducing the required time and quantity of input DNA. In addition, since shearing of the input DNA by sonication is random, sequence biases are not a concern. The primary limitation of immunoprecipita- tion is the quality of antibodies; modified DNA bases share a similar chemical structure with the parent base and each other, making cross-reactivity a significant concern. Furthermore, the resolution of detection is limited by the fragment size; it is not possible to determine where within the 500-base captured fragment the target modification occurred.
An alternative form of DNA immunoprecipitation uses a protein affinity approach instead of antibody; its utility is limited to detecting 5mC with a CpG context. Modified proteins carrying a methyl-binding domain (MBD) are added to the sample in place of the antibody, where they bind to methylated DNA, which is then precipitated. Normally, MBD enrichment uses MBD 2b from MeCP2 (Brinkman et al., 2010), which has a higher affinity for double-stranded, methylated DNA and it is further enhanced by the addition of MBD3L1 (Rauch & Pfeifer, 2010). This technique avoids concerns about antibody specificity and binding sequence (Boch et al., 2009); however, it suffers from the same limitations regarding fragment size, strand specificity, and resolution.