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The principles of DNase I footprinting and its biomedical applications

DNA footprinting technique was developed by David Galas and Albert Schmitz in Geneva in 1977[1]. DNA footprinting is one of the early techniques used to study DNA-protein interactions and is a modification of the Maxam-Gilbert sequencing technique. A DNase I footprinting assay is a DNA footprinting technique in molecular biology/biochemistry. So what is the principle of DNase I footprinting? What biomedical applications are there?

1. What is DNase I footprinting and how does it work?
2. Purpose and application of DNase I footprinting
3. Which type of enzymes is used in DNase I footprinting?
4. What is the difference between DNA fingerprinting and DNA footprinting?

1. What is DNase I footprinting and how does it work?

DNase I footprinting assay is a method of studying DNA-protein interaction and identifying the DNA sequence to which a protein binds[2]. As a lab technique, it is used to find out which segments of DNA molecules are protected by DNA-binding proteins from attack by endonuclease enzymes. The principle of this technique is that DNA-binding proteins protect DNA from external molecules known to cleave or modify DNA.
The mechanism of DNase I footprinting is mainly that proteins bind to DNA fragments, which can protect the phosphodiester bonds of DNA from DNase-catalyzed hydrolysis. After hydrolysis, the DNA fragments are separated on denaturing DNA sequencing gels, and the binding sites can be made by autoradiography. The dots show that the fragments left by DNA molecules after enzyme cleavage are called footprints, and the sequence can be determined by footprints. On autoradiographic pictures of electrophoresis gels, there are no reflectively labeled bands corresponding to the site of protein binding.
The process of DNase I footprinting is as follows:
Clone a piece of DNA that contains the operator site to which the repressor binds. Label one end of the DNA molecules with a radioactive molecule, e. g. radioactive ATP. Digest the DNA with DNase I. DNase I cuts DNA molecules randomly( in contrast to restriction enzymes that cut where they find a particular sequence). This process needs to control the amount of enzyme, it is best to ensure that the adjacent DNA fragments only differ by one nucleotide, and set a control without protein. Choose such gentle conditions that most molecules will be cut only once. The result will be a mixture of radioactive fragments of varying lengths, with the smallest increment in length represented by a single nucleotide. Separate the fragments by electrophoresis. The binding of the lac repressor to the sequence of 24 base pairs in the operator prevents DNase I from attacking that region of the molecule. When the fragments are separated by electrophoresis, those representing the lengths covered by the repressor will be missing from the autoradiogram. The resulting gap is the “footprint”. The same sample of DNA(unprotected by the repressor) is subjected to normal DNA sequencing and the resulting ladder is aligned with the footprint autoradiogram. The exact sequence of bases in the lac operator can then be read directly because they represent the rungs of the ladder missing in the footprint. As shown in figure 1.

Figure 1: Schematic diagram of the DNase I footprint analysis experiment[3]

2. Purpose and application of DNase I footprinting

DNase I footprinting makes it possible to define protein-binding sites on specific DNA molecules. By varying the concentration of DNA-binding proteins, the binding affinity of proteins can also be estimated based on the minimum concentration of the protein that is observed to be footprinted.
An experiment related to DNase I footprinting is the Electrophoretic mobility shift assay (EMSA). EMSA can determine whether a protein binds directly to DNA, while DNase I footprint assay can precisely identify the DNA sequence to which it binds.
EMSA, also known as gel shift assay and gel mobility shift assay, is a special gel electrophoresis technique that uses changes in electrophoretic mobility in vitro to analyze the interaction between DNA and proteins. The program can determine whether a protein or protein mixture is capable of binding to a given DNA or RNA sequence. And the program can sometimes indicate whether more than one protein molecule is involved in the binding complex. The principle of EMSA is the electrophoretic separation of short-term migration of protein-DNA or protein-RNA mixtures in polyacrylamide or agarose gels. The speed at which different molecules and their combinations move through the gel depends on their size and charge and, to a lesser extent, their shape. The control lane, the DNA probe in the absence of protein, will contain a band corresponding to the unbound DNA or RNA fragment. But assuming that the protein can bind to the fragment, there is a clear one where the lane of the bound protein contains another band. The band represents a larger, less mobile complex of the nucleic acid probe bound to the protein, which is in the gel Up "moved up" (because it moves slower).
Under the right conditions, interactions between DNA (or RNA) and proteins are stabilized. Part of this stabilization is due to the "caging effect," because proteins surrounded by the gel matrix cannot diffuse out of the probe until they recombine. The ratio of bound nucleic acid to unbound nucleic acid on the gel is the ratio of free and bound probe molecules when the binding reaction enters the gel. If the initial concentrations of protein and probe, and the stoichiometry of the complex are known, the apparent affinity of the protein for the nucleic acid sequence can be determined. If the protein concentration is unknown and the stoichiometry is complex, the protein concentration can be determined by increasing the concentration of the DNA probe. Until further increasing the DNA probe concentration does not increase the proportion of protein-bound. The number of moles of protein can then be calculated by comparison to a standard dilution of a set of free probes run on the same gel.

3. Which type of enzymes is used in DNase I footprinting?

DNase is a traditional choice because it is a double-stranded endonuclease. The large size of DNase easily creates physical barriers and is easily controlled by the addition of Ethylenediaminetetraacetic acid. And DNase activity is affected by the local DNA structure, so it doesn't cut randomly. DNase I is an endonuclease belonging to DNase. In addition, DNase I is a large molecule, which cannot bind to adjacent DNA-binding proteins due to steric hindrance. To read more about DNase I click on the link.

Table 1: DNase I related products

Product name


Recommended applications

DNase I from bovine pancreas [inquire]


Mainly used in protein research: DNA removal from protein preparations.

Recombinant DNase I (RNase-free)


Ideal for a variety of applications: DNA removal from RNA and protein preparations such as RNase-sensitive cDNA libraries or sample preparation for RT-PCR experiments.

UCF.METM Deoxyribonuclease I (DNase I) GMP-grade


Ideal for a variety of applications: DNA removal from RNA and protein preparations such as RNase-sensitive cDNA libraries or sample preparation for RT-PCR experiments.

4. What is the difference between DNA fingerprinting and DNA footprinting?

DNA fingerprinting is a molecular genetic method that identifies individuals based on unique patterns of DNA. DNA fingerprinting is used by forensic scientists to determine if someone's DNA has been found at a crime scene or elsewhere because everyone has their unique fingerprint unless it's an identical twin. DNA fingerprinting is a molecular biology method that identifies individuals based on their genetic makeup. It was independently developed by Dr. Jeffrey Glassberg in 1983 and the British geneticist Sir Alec Jeffreys in 1984. The original approach of Jeffreys was based on the restriction fragment length polymorphism(RFLP) analysis of minisatellite DNA. Thus, RFLP analysis is one of the main techniques used in DNA fingerprinting. RFLP analysis requires a large amount of DNA, generally more than 25 ng, and this DNA must be fairly intact.
Restriction enzymes cut the DNA in the sample into small fragments in DNA fingerprinting. The digested DNA is then separated by gel electrophoresis and the resulting fragments can be immobilized on membranes by Southern blotting. After that, these fragments can hybridize with the radio-labeled DNA probes containing minisatellites. Oligonucleotide sequences can also be used as probes, and they may directly hybridize with the DNA fragments on the gel. Moreover, the size of the restriction fragments differs depending on the number of repeats of minisatellites, which is unique to an individual. Therefore, the visualization of the fragments allows the identification of the individual.
Unlike DNA fingerprinting, DNA footprinting is a technique researchers use to see where proteins bind to DNA. The DNA with the protein attached is first isolated and digested with varying amounts of DNase, an enzyme that cuts DNA. The DNA is then run on a gel and found where DNase cannot be cut. Where DNase cannot cut is where the protein binds, it looks like a footprint on the gel.

[1] Galas D J, Schmitz A . DNAase footprinting: Simple method for detection of protein-DNA binding specificity[J]. Nucleic Acids Research, 1978, 5(9):3157-3170.
[2] Brenowitz M, Senear D F, Shea M A, et al. Quantitative DNase footprint titration: a method for studying protein-DNA interactions.[J]. Methods in Enzymology, 1986, 130:132-181.
[3]Song C, Zhang S, Huang H. Choosing a suitable method for the identification of replication origins in microbial genomes[J]. Frontiers in Microbiology, 2015, 6:1049.