In Vitro DNase I Footprinting

The association of proteins with the DNA double helix can interfere with the accessibility of the latter to nucleases. This is particularly true when using bulky nucleases such as DNase I. The DNase I footprinting method was developed to take advantage of this fact in the study of DNA-protein interactions: it consists in comparing the pattern of fragments generated by the partial digestion of a DNA sequence in the absence of a protein to that produced by its partial digestion in the presence of said protein. Normally, when the two sets of fragments are separated side by side on a gel, the ladder of DNase I-generated fragments produced in the presence of the protein will feature blank regions (devoid of fragments, indicating protection) and/or enhanced cleavage sites (indicating increased availability to the nuclease). This technique can furthermore reveal if multiple sites for a DNA-binding protein are present on a same fragment and in such a case will also allow the comparison of their respective affinities.

Source: Springer protocols feed by Genetics/Genomics - September 15, 2015 Category: Genetics & Stem Cells Source Type: news