Studying DNA-protein interactions? You Must Know These Techniques

Posted by Ellen Burns on December 3rd, 2020

In many cellular life activities, such as DNA replication, mRNA transcription and modification and viral infection, the interaction between DNA and proteins is involved. In the past half century, predecessors have carried out a lot of exploration in the composition and decomposition process of DNA-protein complexes and developed a series of methodological techniques to study their interaction.

  1. EMSA: Gel Shift Assay

Objective: To detect whether DNA is bound to protein.

Gel shift or electrophoretic mobility shift assay (EMSA) is a special gel electrophoresis technique used to study the interaction between DNA or RNA and proteins in vitro.

In gel electrophoresis, due to the action of electric field, the distance of naked DNA molecules moving to positive electrode is inversely proportional to the logarithm of their molecular weights. If the DNA molecule can bind to the target protein, then its migration in the gel will be blocked due to the increase of molecular weight, so the distance moving towards the positive pole will be correspondingly shortened, so a lagging band will appear in the gel, which is the basic principle of gel retardation experiment.

2. EMSA Upgrade: DNA Competition Experiments

Objective: To detect the exact sequence site of transcription factor protein binding to DNA.

DNA competitive assay is based on traditional EMSA experiments.

An excessive amount of non-labeled competitor DNA is added to the DNA-protein binding reaction system. If the same transcription factor protein binds to the probe DNA, then because the competitive DNA is extremely excessive compared with the probe DNA, most of the transcription factor proteins will be competitively bound, so that the probe DNA is still in a free unbound state, and no blocked band will appear on the autoradiographic picture of the electrophoresis gel.

3. Foot-printing assay

Objective: To detect the exact sequence site of transcription factor protein binding to DNA.

When a segment of DNA molecule binds to specific transcription factors, it can be protected from the cleavage of DNaseI enzyme without producing the corresponding cleavage molecule. As a result, a blank area, commonly known as "footprint", appears on the gel electrophoresis autoradiography picture.

This is actually well understood, taking DNA fragments as positive controls and DNA and protein binding as experimental groups. DNaseI was also allowed to cut randomly. Compared with the experimental group, only the nucleic acid at the binding site will not be shown, that is, the protein and DNA binding region.

4. Methylation interference experiment

Objective: To study the relationship between transcription factors and G residues in DNA binding sites.

Methylationinterference assay is another experimental method designed to study the interaction between protein and DNA based on the principle that DMS (dimethyl sulfate) can methylate the exposed guanine (G) residues in DNA molecules, and hexahydropyridine will specifically cleave the methylated G residues.

This technique can be used to detect the preferential methylation of G residues in target DNA and what effect it will have on the subsequent protein binding, thus revealing the mode of DNA and protein interaction in more detail.

5. Chromatin co-immunoprecipitation technique (ChIP)

Objective: To study the sequence information of transcription factor and DNA binding site.

The combination of CHIP with other methods has expanded its scope of application: the CHIP-on-chip method established by the combination of CHIP and gene chip has been widely used for high-throughput screening of specific trans-factor target genes; RNA-CHIP is used to study the role of RNA in gene expression regulation.

6. Southwestern Hybridization

Objective: To study the nature of DNA binding protein (molecular weight).

DNA-protein blot hybridization, or Southwestern blot, has been widely used in many fields of molecular genetics and molecular biology since its establishment in 1980 by Bowen et al. The crude nucleoprotein extract was analyzed by SDS-PAGE electrophoresis, and bound to isotope-labeled specific DNA sequence probes after transfer. Site-specific DNA-binding proteins bind DNA probes by means of hydrogen bonds, ion bonds, and hydrophobic bonds, so that DNA-binding proteins can be qualitatively and quantitatively analyzed by autoradiography.

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Ellen Burns

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Ellen Burns
Joined: November 1st, 2019
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