A DNase footprinting assay[1] is a DNA footprinting technique from molecular biology Molecular biology is the study of biology at a molecular level. The field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interactions between DNA, RNA and protein/biochemistry Biochemistry is the study of the chemical processes in living organisms. It deals with the structure and function of cellular components such as proteins, carbohydrates, lipids, nucleic acids and other biomolecules that detects DNA Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information. DNA is often compared to a set of blueprints or a recipe, or a code, since it contains the instructions needed-protein Proteins are organic compounds made of amino acids arranged in a linear chain and folded into a globular form. The amino acids in a polymer chain are joined together by the peptide bonds between the carboxyl and amino groups of adjacent amino acid residues. The sequence of amino acids in a protein is defined by the sequence of a gene, which is interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. This makes it possible to locate a protein binding site on a particular DNA molecule. The method uses an enzyme, deoxyribonuclease A deoxyribonuclease is any enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. Deoxyribonucleases are thus one type of nuclease. A wide variety of deoxyribonucleases are known, which differ in their substrate specificities, chemical mechanisms, and biological functions (DNase, for short) to cut the radioactively end-labelled DNA, followed by gel electrophoresis Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid , ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix. DNA Gel electrophoresis is generally only used after amplification of DNA via PCR. It is usually performed for analytical purposes, but may be used as a preparative to detect the resulting cleavage pattern.
For example, the DNA fragment of interest may be PCR In molecular biology, the polymerase chain reaction is a technique to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA amplified using a 32P 5' labeled primer, with the end result being many DNA molecules with a radioactive label on one end of one strand of each double stranded molecule. Cleavage by DNase will produce fragments, the smaller of which will move further on the electrophoretic gel. The fragments which are smaller with respect to the 32P-labelled end will appear further on the gel than the longer fragments. The gel is then used to expose a special photographic film.
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 DNA, the binding site is protected from enzymatic cleavage. This protection will result in a clear area on the gel which is referred to as the "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.
This technique was developed by David Galas and Albert Schmitz at Geneva in 1977[2]
References
- ^ Brenowitz M, Senear DF, Shea MA, Ackers GK (1986). "Quantitative DNase footprint titration: a method for studying protein-DNA interactions". Methods in Enzymology 130: 132–81. PMID A PMID is a unique number assigned to each PubMed citation of life sciences and biomedical scientific journal articles. The related Pubmed Central archive may additionally assign a separate number, a PMCID (PubMed Central Identifier), normally written with a PMC prefix 3773731.
- ^ Galas DJ, Schmitz A (Sep 1978). "DNAse footprinting: a simple method for the detection of protein-DNA binding specificity". Nucleic acids research 5 (9): 3157–70. PMID A PMID is a unique number assigned to each PubMed citation of life sciences and biomedical scientific journal articles. The related Pubmed Central archive may additionally assign a separate number, a PMCID (PubMed Central Identifier), normally written with a PMC prefix 212715. PMC PubMed Central is a free digital database of full-text scientific literature in biomedical and life sciences 342238. http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=212715.
[[de:DNase Footprinting Assay]
Categories: Molecular biology Molecular biology is the study of biology at a molecular level. The field overlaps with other areas of biology, particularly genetics and biochemistry. Molecular biology chiefly concerns itself with understanding the interactions between the various systems of a cell, including the interrelationship of DNA, RNA and protein synthesis and learning | Laboratory techniques Laboratory techniques, as used in Biology, Biochemistry, Chemistry, Molecular biology, etc |
