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The Experimental Techniques for Investigating Protein-dna Interaction

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The experimental techniques for investigating protein-DNA interaction are classified in two forms, i.e. in vitro and in vivo. The studies of in vivo are helpful because of the preservation of the natural structure of interaction sides. However, in multi-proteins, it is hard to figure out which part of protein is directly connected to DNA and protects it. In contrast, the study of in vitro is suitable on purified proteins and subunits of protein.

At the end of the 1960s, the first studies were carried out on DNA-protein interaction . Nitrocellulose filter binding assays was the first offered technique for this purpose . In the 1990s, many diverse experimental techniques have been developed for studying DNA-protein interaction. To days many techniques are available for the detection and characterization of protein-nucleic acid complexes and most have advantages and disadvantages. Such techniques including but not limited to Electrophoretic Mobility Shift Assays (EMSA) (Fried 1989), DNase I Footprinting (Brenowitz et al. 1986), Bacterial One-hybrid System and techniques based on Chromatin immunoprecipitation analysis (ChIP) e.g. chromatin immunoprecipitation with DNA microarray (ChIP-chip; Lieb et al. 2001), chromatin immunoprecipitation-sequencing (ChIP-Seq; Johnson et al. 2007), ChIP-exo (Pugh 2012) and chromatin immunoprecipitation-Paired-end tags (ChIP-PET; Wu et al.2013) have been used. Many techniques are available for the detection and characterization of protein-nucleic acid complexes and most have advantages and disadvantages determination and characterization DNA-protein relation.Electrophoretic Mobility Shift Assays (EMSA); Electrophoretic Mobility Shift Assays (also known as “band shift assays” and “mobility shift electrophoresis”) has a standard protocol for investigating a wide range of nucleic acid–protein interactions from single protein-binding events to assembly of large complexes such as the spliceosome (Malloy 2000; Rio 2014). EMSA technique has been originally introduced by Fried 1989 and nowadays many variants have been described in the literatures. EMSA is a simple, quick, and very sensitive laboratory technique for testing nucleic acid/protein specific interaction qualitatively, although, under appropriate condition are used for quantitative purpose.

However, EMSA is not without limitations and more important limitations and problems were encountered (Hellman and Fried 2007). This technique is based on the observation that the segments of binding nucleic acid to protein cause a decrease in the segment’s electrophoretic mobility compared with the free nucleic acid in agarose gel under native condition or non-denaturing polyacrylamide gel (Vinckevicius and Chakravarti, 2012; Rio 2014). In this technique crude protein mixture or purified proteins are mixed with the nucleic acid sequence in a suitable buffer and specific binding is allowed to occur, stable complexes of nucleic acid and protein (the probe may be bound in nonspecific manner by other proteins) were separated by nondenaturing gel electrophoresis; not only for study of nucleic acid sequence requirements of binding but also diverse aspect of nucleic acid-protein interaction including but not limited to, kinetics of binding (such as affinity constants), identification and characterization of binding proteins, and cofactor requirements. A wide variety of nucleic acid and protein lengths (lengths from short oligonucleotides/amino acid to several thousand) and distinct nucleic acid structures (single-stranded, duplex, triplex and quadruplex nucleic acids as well as small circular DNAs) are compatible with EMSA technique (Hellman and Fried 2007). However, with this technique, the range of DNA sequencing binding site is obtained, it does not have enough precision in determining the precise site and the cooperative interaction elements (for more detail see Hellman and Fried 2007).

Deoxyribonuclease I (DNaseI) Footprinting; A valuable technique not only for identifying but also for characterizing DNA-protein interactions is DNaseI Footprint method (Carey et al. 2013). This method is also used for sequence-selective recognition of DNA-binding ligands (Hampshire et al. 2007). The concept is that the sequence-selective binding protein protects the phosphodiester backbone of DNA in and around its binding site from DNase I-catalyzed hydrolysis thus generates a “footprint” in the cleavage ladder. DNase I which non-specifically cuts a single strand of a double-stranded DNA helix that is not protected by e.g. binding protein, is a convenient endonuclease for detecting and locating the position of sequence selective binding proteins (Bailly et al., 2015). The binding sites visualized by the autoradiography of DNA segments produced by electrophoresis on denaturing DNA sequencing gels (Brenozwitz et al. 2001; Vinckevicius and Chakravarti, 2012). steric hindrance arising from DNA- bounded protein in the site and its adjacent do not allow DNaseI to bind directly to the binding site and 8-10 base pairs around it (Carey et al. 2013).

Despite the great value of the above-mentioned techniques, the importance of the performance of binding proteins to these techniques is not recognizable. One solution, for this purpose, is the use of reporter assays. Examples of it are the genes of Chloramphenicol acetyltransferase, green fluorescent protein and assay-based luciferase. In these techniques, the region of transcription factors in the upstream region of a cloned reporter assay and its transcription activity is measured. Mutations in transcription factors or their binding site make the analysis of the sequences or important elements possible in interaction. A different technique based on reporter assay is the formation of mono-hybrid and bi-hybrid systems in bacteria that causes the revealing of transcription in genes because of the interaction of many binding proteins to specific regions of DNA.

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The experimental techniques for investigating protein-DNA interaction. (2018, December 17). GradesFixer. Retrieved June 29, 2022, from https://gradesfixer.com/free-essay-examples/the-experimental-techniques-for-investigating-protein-dna-interaction/
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