I. Introduction
In many cell life activities, such as DNA replication, mRNA transcription and modification, and viral infection, etc., the problem of interaction between DNA and protein is involved.
Many important genes have been isolated from the development of recombinant DNA technology. The key issue now is to reveal how environmental factors and developmental signals control the transcriptional activity of genes. To do this you need:
a. Identification and analysis of DNA elements involved in the regulation of gene expression;
b. isolating and identifying protein factors to which these cis-elements specifically bind;
Research on these issues involves the interaction between DNA and proteins .
Experimental methods for studying DNA-protein interactions mainly include:
a, gel retardation experiment;
b, DNase 1 footprint test;
c, methylation interference experiment;
d, in vivo footprint experiment;
e. Pull down the experiment.
Second, gel retardation experiment
1 concept
The gel retardation assay, called the DNA mobility shift assay or the band retardation assay, was developed in the early 1980s to study DNA in vitro. A special gel electrophoresis technique for protein interactions.
2 Principle
In gel electrophoresis, the distance traveled by a bare DNA molecule to a positive electrode is inversely proportional to the logarithm of its molecular weight due to the action of an electric field. If a DNA molecule binds to a specific protein, its migration in the gel will be retarded due to the increase in molecular weight, so the distance moved toward the positive electrode will be correspondingly shortened, thus being in the gel. The lag band appears, which is the basic principle of the gel retardation experiment.
3 process
First prepare a cellular protein extract (theoretically contains a specific transcription factor)
Labeling the DNA fragment to be detected with a radioisotope (binding site containing transcription factors)
The labeled probe DNA is incubated with the cellular protein extract to produce a DNA-protein complex
Non-denaturing polyacrylamide gel electrophoresis under conditions that control the DNA-protein to remain bound
Finally, autoradiography was performed to analyze the electrophoresis results.
4. Analysis of experimental results:
a. If radiolabeled bands are concentrated at the bottom of the gel, this indicates that there is no transcription factor protein in the cell extract that can bind to the probe DNA;
b. If a radiolabeled band is present at the top of the gel, this indicates that the cell extract has a transcription factor protein that binds to the probe DNA.
5. DNA competition experiment:
The specific practices of the DNA competitive assay are as follows:
Excessive unlabeled competitor DNA is added to the DNA-protein-bound reaction system. If it binds to the same transcription factor protein as the probe DNA, then the competitive DNA is compared with the probe DNA. Extremely large, so that most of the transcription factor proteins will be competitively bound, so that the probe DNA is still in a free unbound state, and it can be blocked on the autoradiogram of the electrophoresis gel. Bands;
If the competitive DNA added to the reaction system does not compete with the probe DNA for binding to the same transcription factor, a band of blockage will appear on the autoradiograph image in the electrophoresis gel.
6, application:
a, gel retardation experiments can be used to identify the presence of transcription factor proteins that bind to a particular DNA (containing a transcription factor binding site) in a particular type of cellular protein extract;
b, DNA competition experiments can be used to detect the exact sequence of the transcription factor protein binding to DNA;
c. The effect of the competition of the mutation and the binding of the transcription factor can be studied by competing for the base mutation of the transcription factor binding site in the DNA;
d. It is also possible to isolate specific transcription factors by affinity chromatography using the binding of DNA to specific transcription factors.
Third, the footprint experiment
1. Definition:
A foot-printing assay is a specialized experimental method for detecting the position of a DNA sequence specifically bound by a specific transcription factor protein and its nucleotide sequence structure.
2, the principle:
When a segment of the DNA molecule binds to a specific transcription factor, it can be protected from the cleavage of the DNaseI enzyme without producing the corresponding cleavage molecule. The result is on the gel electrophoresis autoradiography image. A blank area appeared, commonly known as the "footprint."
3. Process:
Double stranded DNA molecule to be tested in vitro for 5 'end-labeled with 32 P, and cut with the appropriate restriction enzyme in which an end, so they obtained single-stranded end-labeled double-stranded DNA
In vitro, mixed with cellular protein extracts (nuclear extracts) to form DNA-protein complexes
A small amount of DNase I was added to the reaction mixture, and the amount was controlled to reach an average of each DNA strand, and only one break of the phosphodiester bond occurred:
a. If there is no specific protein bound to DNA in the protein extract, after DNase I digestion, it will produce 1 nucleotide, 2 nucleotides, 3 nucleotides from the end of the radioactive label--- --- and so on a series of uninterrupted continuous DNA fragment gradient populations whose front and back lengths differ by one nucleotide;
b. If the DNA molecule binds to a transcription factor in the protein extract, the DNA of the bound site can be protected from the degradation of the DNase I enzyme to remove the protein, and the sample is electrophoresed on a 20% sequence gel. In two groups:
a, experimental group: DNA + protein mixture
b, control group: only DNA, not incubated with protein extract
Finally, autoradiography was performed to analyze the experimental results.
4. Judgment of results:
In the experimental group, the sequence indicated by gel electrophoresis showed that the blank region indicated that it was the protein binding portion of the transcription factor; compared with the control sequence, the corresponding nucleotide sequence of the DNA segment of the protein binding site was obtained.
5. Other footprint test methods:
In addition to the DNase1 footprint test, several other types of footprint experiments have been developed, such as:
a, free hydroxyl footprint test; b, phenanthroline copper footprint test; c, DMS (dimethyl sulfate) footprint test
Principle of DMS (Dimethyl Sulfate) Footprint Experiment
DMS is capable of methylating the naked guanine (G) residue in the DNA molecule, which in turn specifically cleaves the methylated G residue. If a segment of the DNA molecule binds to a transcription factor, methylation of the G residue can be avoided from the cleavage of the hexahydropyridine.
Fourth, methylation interference experiment
1. Concept:
The Methylation interference assay is based on the fact that DMS (dimethyl sulphate) methylates the guanine (G) residues exposed in the DNA molecule, and the hexahydropyridine is methylated. Another experimental method for studying the interaction of proteins with DNA is based on the principle of specific chemical cleavage.
This technique can be used to detect the preferential methylation of G residues in the target DNA, and what effect will it have on the subsequent protein binding, thus revealing in more detail the pattern of DNA-protein interaction.
2. Experimental steps:
Target DNA is treated with DMS to localize methylation (on average, only one G base methylation per DNA)
Incubate with cellular protein extracts to promote DNA and protein binding
Gel electrophoresis was performed to form two target DNA bands:
a, a normal electrophoresis band of DNA without protein binding
b. DNA electrophoresis bands that exhibit hysteresis when combined with specific proteins
The two DNA bands were cut out from the gel and cut with hexahydropyridine. The results were:
a. The methylated G residue is cleaved: since the transcription factor protein can only bind to the normal binding site where methylation has not occurred, the G residue in the transcription factor DNA binding site sequence is DMS A. After the basement, the transcription factor cannot bind to its binding site (cis element), so that the G residue in the binding site is also cleaved by the hexahydropyridine;
b. Target DNA sequences without methylated G residues will not be cleaved
The DNA band that binds the protein and the DNA band that does not bind to the protein are cleaved by the hexahydropyridine and then subjected to gel electrophoresis.
Autoradiography, reading and analyzing the results
3. Judgment of results:
a, the target DNA sequence that binds to the transcription factor protein, after being cleaved by the hexahydropyridine, the electrophoretic separation presents two bands with a blank area
b. The target DNA sequence bound by different transcription factor proteins is cleaved by hexahydropyridine, and three bands are formed by electrophoresis separation, and no blank region appears.
4. Application:
a, methylation interference experiments can be used to study the relationship between transcription factors and G residues in the DNA binding site;
b. It is an effective supplement to the footprint test to identify the precise location of DNA-protein interactions in footprint experiments.
5, disadvantages:
DMS can only methylate the G and A residues in the DNA sequence without methylating the T and C residues.
V. In vivo footprint experiment
One of the common shortcomings of the three methods discussed above for studying the interaction of transcription factors with DNA is that they are experiments conducted in vitro, so one considers whether these experimental results reflect the real life processes occurring within the cell. That is, the actual interaction between DNA and protein that occurs within the cell.
To answer this question, the scientists designed an in vivo foot-printing assay, which can be seen as a variant of the in vitro DMS footprint experiment. 1, the principle:
The principle of the in vivo footprint test is in principle essentially indistinguishable from the in vitro DMS footprint test, ie
a, DMS can methylate the G residue;
b, hexahydropyridine can specifically cleave methylated G residues;
c. The G residue in the recognition sequence that binds to the specific transcription factor protein is not protected by the protein and is not methylated by DMS, so it is not cleaved by the dihydropyridine;
d. Comparing with the sequence ladder formed by the naked DNA of the control, it is found that the sequence ladder formed by living cell DNA lacks the corresponding band in which the G residue is not cleaved.
2. Process:
Treatment of intact free cells with a limited amount of chemical reagent DMS allows the concentration of DMS that penetrates into the cell to just cause methylation of the G residue of the native chromosomal DNA
DNA was extracted from these DMS-treated cells and hexahydropyridine was added in vitro for digestion.
After PCR amplification, gel electrophoresis analysis is performed because the number of cloned DNA fragments is sufficient in in vitro experiments, and any specific DNA isolated from chromosomal DNA is used in the in vivo footprint test. The quantity is negligible, so PCR amplification is required to obtain a sufficient amount of specific DNA.
Autoradiography, reading and recording the results of the reading
3. Judgment of results:
a DNA segment capable of binding to a transcription factor protein wherein the G residue is protected and thus not methylated by DMS to avoid cleavage of the hexahydropyridine;
b. On the naked DNA molecule in vitro, the G residue is methylated by DMS and cleaved by hexahydropyridine.
6. Pull-down assay
Pull-down experiments, also known as binding assays in vitro, are a method of detecting protein interactions in test tubes. The basic principle is that a gene encoding a small peptide (for example, biotin, 6-His tag, and glutathione transferase) is recombined with a gene encoding a bait protein and expressed as a fusion protein. The fusion protein is isolated and purified and bound to the magnetic beads to be solid-phased, and then mixed with the cell extract expressing the protein of interest for a suitable period of time, for example, at 4 ° C overnight, so that the target protein is immobilized on the surface of the magnetic beads. The bait protein in the fusion protein is fully bound. The target protein bound to the bait protein in the immobilized fusion protein (ie, the fusion protein bound to the magnetic beads) is collected by centrifugation, and the target protein is separated from the bait protein by boiling treatment to obtain a solid phase support (for example, magnetic beads). The detachment is carried out, the sample is collected, and the antibody of the target protein is subjected to Western blotting analysis to detect the target protein of the target with the bait protein.
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