DNase I Footprinting Assay Service

Profacgen provides DNase I footprinting assay service for the detection of DNA-protein interaction and identification of the exact binding sites of DNA-binding proteins. DNase I footprinting assay is based on the fact that a DNA-binding protein often protects its bound DNA fragment from enzymatic cleavage, making it possible to locate a protein binding site on a particular DNA fragment.

This method uses DNase I to digest the radioactively-labeled or fluorescently-labeled DNA fragment, and then uses gel electrophoresis to detect the resulting cleavage pattern. The cleavage pattern of the DNA in the absence of a DNA binding protein, typically referred to as free DNA, is compared to the cleavage pattern of DNA in the presence of a DNA binding protein. If the protein binds to the DNA fragment, the binding site is protected from enzymatic cleavage. This protection will result in a clear area on the gel which is referred to as a “footprint”. By varying the concentration of the DNA-binding protein, the binding affinity of the protein can be estimated according to the minimum concentration of protein at which a footprint is observed.

Schematic Diagram of the DNase I Footprinting Assay

Classic DNase I footprinting assay labels DNA molecules with radioisotope, and need to run sequencing gel for detection. However, as radioactive labeling has safety concern and is not as convenient as fluorescence labeling, Profacgen uses fluorescence labeling in combination with sequencer (e.g. supplied by Applied Biosystem), enabling our DNase I footprinting assay safer, faster and more sensitive.

As DNase I footprinting assay is used to determine the precise DNA sequence bound by the protein of interest, it is recommended to be performed after interaction confirmation by Electrophoretic Mobility Shift Assay (EMSA). Therefore, in addition to purified protein and the DNA fragment, EMSA data and relating experimental parameters will all help design the DNase I footprinting assay.

Experiment flowchart:

Protein and nucleotide preparation (optional)

EMSA verification (optional)

DNase I footprinting

Data analysis

Result delivery

Please feel free to contact us for more information of our professional DNase I footprinting assay service.

References from our customer:

Gonzalez, Martín, et al. "SetRICE391, a negative transcriptional regulator of the integrating conjugative element 391 mutagenic response." DNA repair 73 (2019): 99-109.
This study provides evidence indicating that SetRICE391 acts as a transcriptional repressor of the ICE391-encoded mutagenic response. The DNase I footprint analysis was undertaken as a custom contract service by scientists at Profacgen.