In 1978, David J. Galas and Albert Schmitz developed the DNA footprinting technique to study the binding specificity of the lac repressor protein.
[2] Polymerase chain reaction (PCR) amplify and label region of interest that contains a potential protein-binding site, ideally amplicon is between 50 and 200 base pairs in length.
The portion of DNA template without protein will be cut at random locations, and thus when it is run on a gel, will produce a ladder-like distribution.
Note: Maxam-Gilbert chemical DNA sequencing can be run alongside the samples on the polyacrylamide gel to allow the prediction of the exact location of ligand binding site.
Radioactivity has been traditionally used to label DNA fragments for footprinting analysis, as the method was originally developed from the Maxam-Gilbert chemical sequencing technique.
Electrophoretic sequencing gels or capillary electrophoresis have been successful in analyzing footprinting of fluorescent tagged fragments.
[4] Ultraviolet irradiation can be used to excite nucleic acids and create photoreactions, which results in damaged bases in the DNA strand.
The workflow for this method has an additional step, once both your protected and unprotected DNA have been treated, there is subsequent primer extension of the cleaved products.
[6][7] The extension will terminate upon reaching a damaged base, and thus when the PCR products are run side-by-side on a gel; the protected sample will show an additional band where the DNA was crosslinked with a bound protein.
A disadvantage is that the gel can be difficult to interpret, as the bound protein does not protect the DNA, it merely alters the photoreactions in the vicinity.
A region of interest is amplified between the linker and a gene-specific primer, and when run on a polyacrylamide gel, will have a footprint where a protein was bound.
If the DNA fragment to be analyzed is produced by polymerase chain reaction (PCR), it is straightforward to couple a fluorescent molecule such as carboxyfluorescein (FAM) to the primers.
Open chromatin assays such as DNase-Seq[13] and FAIRE-Seq[14] have proven to provide a robust regulatory landscape for many cell types.
[18] Site-centric methods, on the other hand, find footprints given the open chromatin profile around motif-predicted binding sites, i.e., regulatory regions predicted using DNA-protein sequence information (encoded in structures such as position weight matrix).