DNase footprinting assay

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DNase footprinting assay ( DNase footprint assay ) is a molecular biological method for the determination of protein-DNA interactions .

principle

The assay is based on the fact that DNA is to some extent protected from enzymatic cleavage at sites where a protein is bound . The enzyme deoxyribonuclease ( DNase for short ) is used in the process.

application

The method allows binding sites of proteins on a DNA molecule, e.g. B. exactly to map the promoter of a protein-coding gene in vitro . The proteins are specifically generated beforehand using molecular biological methods or they come from cell or nuclear extracts . When using pure proteins, the assay allows quantitative statements about the binding. The method was developed by David Galas and Albert Schmitz in Geneva.

method

The investigated DNA chains of a few hundred base pairs in length are radioactively marked at one end of one of the two strands . They are mixed with protein extract or a sample of specifically selected proteins. Under certain circumstances, specific non-covalent DNA-protein bonds are formed which are based on hydrophobic effects , ionic bonds and hydrogen bonds . As in the original publication by Galas and Schmitz, DNase I from bovine pancreas is usually used. Then the DNA is separated from the proteins. During digestion, it is ensured that DNA chains are split once by the enzyme or at a few points. This results in fragments of DNA of various lengths. Those fragments that contain the marked end are of particular interest, because only these are visible in the autoradiography after the subsequent denaturing polyacrylamide gel electrophoresis . In gel electrophoresis, the control without protein addition shows an almost continuous distribution of the fragments, since the speed of a DNA fragment is proportional to its length. In the reactions with protein addition, DNase cannot act on sections where proteins bind. DNA fragments whose length falls within this binding range are missing, which is visible through a gap in the continuous spread in the gel. Such a gap is known as the protein's footprint on DNA. The binding sequence of the protein on the DNA is mapped with nucleotide accuracy using the sequence reactions of the same fragment applied in parallel using the method of Maxam and Gilbert .

classification

Advantages of the DNase Footprinting Assay are the exact mapping, the possibility to examine several binding sites on a DNA and to quantify the binding strength. Disadvantages are the use of radioactivity, mostly in the form of the phosphorus isotope 32 P, as well as the greater experimental effort compared to alternative methods such as the filter binding test and the electrophoretic mobility shift assay . There are variations of the method using other nucleases and chemicals such as dimethyl sulfate , which cause DNA breaks.

Individual evidence

  1. ↑ Michael Brenowitz, Donald F. Senear, Madeline A. Shea, Gary K. Ackers: Quantitative DNase footprint titration: a method for studying protein-DNA interactions. In: Methods in Enzymology . Vol. 130, 1986, ISSN  0076-6879 , pp. 132-181, PMID 3773731 , doi : 10.1016 / 0076-6879 (86) 30011-9
  2. ^ A b D. J. Galas and A. Schmitz: DNAse footprinting: a simple method for the detection of protein-DNA binding specificity. In: Nucleic Acids Research . Vol. 5, No. 9, 1978, pp. 3157-3170, PMID 212715