Figure 1. ChIP-chip. (a) Types of DNAs used in ChIP-chip. Oligonucleotides tiling genomic regions (left); PCR-amplified genomic regions (right). (b) ChIP-chip experimental design. A large variety of protein–DNA and protein–protein crosslinks are created nonspecifically, owing to the nonspecific nature of formaldehyde crosslinking. An antibody (orange) either specific for the protein of interest (blue) or specific for an epitope tag fused to the protein of interest is used in immunoprecipitation (IP) in the experimental sample. This IP will enrich for the target protein, including protein directly bound to genomic DNA-binding sites, and also for protein indirectly associated with DNA via protein–protein interactions. A control (‘mock’) IP is performed using either no antibody, an irrelevant antibody or pre-immune IgG antibodies. This mock IP is not expected to enrich for the target protein of interest.

Figure 2. DamID. (a) Types of DNAs used in DamID microarray hybridizations. Oligonucleotides tiling genomic regions (left); cDNAs or PCR-amplified genomic regions (right). (b) DamID experimental design. The protein of interest (blue) is overexpressed in vivo from a plasmid as a fusion to Dam (purple). Wherever the protein binds DNA, Dam will methylate adenines within GATC sites in the vicinity of the binding sites. The methylated sites are digested with the methyl-specific restriction enzyme DpnI, which cuts only at methylated GATC sites. The smaller DpnI digestion fragments, corresponding to the methylated regions, are either purified by sucrose gradient centrifugation or specifically amplified using a methylation-specific PCR protocol. (Panel (b) was adapted from [7] and [51] with permission by Macmillan Publishers Ltd.).

Figure 3. Protein binding microarrays (PBMs). (a) Types of DNAs used in PBMs. Short double-stranded oligonucleotides created by primer extension using a universal primer (red), complementary to a sequence present on spots either on an oligonucleotide array [8] or in solution [^{9 and 10••}] (left); regions of short double-stranded DNA created by self-hairpinning oligonucleotides on an oligonucleotide array [50^••] (center); longer double-stranded DNAs resulting from PCR amplification of genomic regions [10^••] (right). (b) PBM experimental design. Double-stranded DNA microarrays can be either bound by an epitope-tagged TF (blue) and labeled by a fluorophore-conjugated (green) antibody specific for the tag [10^••] (left), or bound by a directly fluorophore-labeled TF [50^••] (right). (Panel (b) was adapted from [10^••] with permission from Nature Publishing Group.).

Comparison of ChIP-chip, DamID and PBM technologies