DNA footprinting

DNA footprinting is a method of in vitro DNA analysis that assists researchers in determining transcription factor (TF) associated binding proteins. This technique can be used to study protein-DNA interactions both outside and within cells.

Transcription factors are regulatory proteins that assist with various levels of DNA regulation. These regulatory molecules and associated proteins bind promoters, enhancers, or silencers to drive or repress transcription and are fundamental to understanding the unique regulation of individual genes within the genome.

First developed in 1978, primary investigators David J. Galas, Ph.D. and Albert Schmitz, Ph.D. modified the pre-existing Maxam-Gilbert chemical sequencing technique to bind specifically to the lac repressor protein. Since the technique's discovery, scientific researchers have developed this technique to map chromatin and have greatly reduced technical requirements to perform the footprinting method.

The most common method of DNA footprinting is DNase-sequencing. DNase-sequencing uses DNase I endonuclease to cleave DNA for analysis. The process of DNA footprinting begins with polymerase chain reaction (PCR) to increase the amount of DNA present. This is to ensure the sample contains sufficient amount of DNA for analysis. Once added, proteins of interest will bind to DNA at their respective binding sites. This is then followed by cleavage with an enzyme like DNase I that will cleave unbound regions of DNA and keep protein-bound DNA intact. The resulting DNA fragments will be separated using Polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis allows researchers to determine fragment sizes of the protein-bound DNA fragments that have since been cleaved. This is indicated by the gap regions on the gel, areas where there are no bands, representing specific DNA-protein interactions.