Plasmid DNA is a small double stranded circular DNA molecule that is exposed (without protein binding), structurally simple, independent of bacterial nucleoid DNA, and has the ability to self replicate. Image plasmids usually carry only a small number of genes, especially those related to antibiotic resistance; In addition, plasmids can be transmitted between different bacterial cells because plasmid molecules have a genetic structure with replication function, which can carry some genetic information. Moreover, their self replication ability and ability to carry genetic information are very strong. After gene expression, plasmids can make their host cells exhibit corresponding traits. Therefore, plasmids are often used as gene carriers and are widely used in disciplines such as molecular biology, biochemistry, and cell biology.
1、 The morphology of plasmids
It is generally imagined that plasmid DNA appears to be a simple circular structure, but in reality, the molecules of double stranded circular DNA undergo axial deformation or double helix distortion. Axial deformation leads to regular spatial deformation of the helix axis, resulting in topological structures. Moreover, due to the different actual number of base pairs in each helix turn of closed circular DNA, various super helix shapes of plasmid DNA are formed.
A normal plasmid is double stranded DNA, but during the plasmid extraction process, due to mechanical forces, pH, reagents, and other factors, the plasmid DNA strand may break, open loop, or spiral from open loop to linear. Most plasmid extracts contain plasmids in three different configurations.
Covalent closed circular DNA (scDNA), where two strands of the plasmid remain unbroken and two helices are cross-linked, distorting to form a super helix structure based on the helix of DNA. Open loop DNA (ocDNA), where one strand in the double strand breaks, causes the helix of the supercoiled DNA to relax and become a circular plasmid. Linear DNA, in which both strands of a plasmid break and become linear molecules.
On the basis of the basic configuration, the three plasmids each have multiple polymerization forms.
In addition, plasmids may appear in oligomeric form, such as tandem, which may exist in different isomeric forms.
All these forms of plasmid can be observed by agarose gel electrophoresis, but the number of bands will vary depending on your plasmid extraction technology, the freshness of the plasmid, the configuration of the electrophoresis solution, and the configuration of agarose gel.
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1. Electrophoretic migration rates of three plasmid forms
The three types of plasmids have different shapes when unfolded. During the electrophoresis process, plasmids will form three bands. Open loop plasmids have the largest unfolded area and resistance, appearing at the end. After electrophoresis, the comparison marker is usually larger than the actual size, and the linear one will be in the middle. After electrophoresis, the comparison marker is equal to the actual size, while the super spiral structure will run at the front due to its compact shape and small volume. After electrophoresis, the comparison marker is smaller than the actual size; Due to the limited linear morphology, the size of plasmid electrophoresis is generally not equal to the actual size of the plasmid.
The image plasmid does not have a clear pattern during replication and may be in multiple replication stages, so the extracted plasmid may have more than or less than three bands during electrophoresis detection. We can determine whether multiple bands are all target plasmids by performing restriction endonucleases to validate the plasmids. If only the expected size band is present during electrophoresis of the digested product, it indicates that different bands are all target plasmids, but with different shapes.
2. Analysis of supercoiled plasmids
1) Qualitative and quantitative analysis
Using a nuclease with special functions (which has double stranded DNA specific exonuclease activity and single stranded DNA endonuclease activity, can degrade genomes, linear and open-loop plasmids, but cannot degrade supercoiled double stranded DNA) to process plasmids, the open-loop and linear bands will be digested, leaving only supercoiled bands. Compared with untreated plasmids, the position and proportion of supercoiled bands can be easily and quickly determined, that is, the qualitative and quantitative analysis of plasmids with supercoiled configurations has been carried out.
2) Analysis of Super Spiral Polymers
Partial restriction endonuclease cleavage experiment: Plasmids are taken for restriction endonuclease cleavage. As the supercoiled monomer plasmid only has one cleavage site, while the supercoiled dimer or polymer has two or more identical cleavage sites. As the enzyme amount and reaction time increase, a phenomenon occurs during plasmid single enzyme cleavage: when the enzyme amount is small, only one cleavage site of the supercoiled dimer or polymer is cleaved, and linear bands of two or more times the size will be seen on the electrophoresis map. When the enzyme amount increases or the reaction time increases, two or more cleavage sites of the supercoiled dimer or polymer are cut open, and only plasmid sized bands will be seen in the electrophoresis map. This method can confirm the electrophoretic map position of plasmid oligomers.
2、 Extraction of plasmids
The extraction of plasmids involves removing RNA, separating plasmids from bacterial genomic DNA, removing proteins and other impurities, in order to obtain relatively pure plasmids.
There are many methods for extracting plasmid DNA, including alkaline lysis, boiling method, SDS lysis method, and organic solvent method. Regardless of which method is used to extract plasmids from bacteria, the main steps include three steps: collecting and cracking bacteria, releasing DNA from cells, and isolating and purifying plasmid DNA.
1. Alkali cracking method
1) Bacterial cell wall
The cell wall of Gram positive bacteria: The cell wall of G+bacteria has a thick (20-80nm) and dense peptidoglycan layer, up to 50 layers, accounting for 40% to 95% of the cell wall composition. It is closely connected to the outer layer of the cell membrane. Some G+bacteria contain phosphonic acid in their cell walls, which is a polymer of glycerol and ribol. phosphonic acid is usually present as an ester of sugar or amino acid.
The cell wall of Gram negative bacteria: G-bacterial cell wall is thinner than G+bacterial cell wall (15-20nm) and has a more complex structure, consisting of an outer membrane and a peptidoglycan layer (2-3nm). There is a clear space between the cell wall and the cytoplasmic membrane, called the parietal gap. The basic component of the outer membrane of G-bacterial cell wall is lipopolysaccharide (LPS), which, like the cytoplasmic membrane, is also a bilayer lipid, but contains polysaccharides and proteins in addition to phospholipids.
The main difference between Gram positive/negative bacteria is that G+bacteria have strong mechanical strength, but their cell walls are easily digested by lysozyme, while G – bacteria have weak cell wall strength but are tolerant to lysozyme. EDTA is mainly used to destroy the cell wall of Gram negative bacteria: The outer membrane structure of G-bacteria is usually maintained by the combination of divalent cations Ca2+or Mg2+with lipopolysaccharides and proteins. Once EDTA chelates Ca2+or Mg2+, a large number of lipopolysaccharide molecules will fall off, causing holes to appear in the outer membrane of the cell wall.
2) Cracking
(1) Solution I: 50mM glucose; 25mM Tris Cl; 10mM EDTA; PH 8.0.
Tris Cl: The pH of a stable solution.
50mM glucose: Keep the suspended bacterial body in a suspended state, so that it does not quickly deposit to the bottom of the tube; Facilitate sufficient lysis of bacterial cells. Non essential cracking components, removal has no essential impact on extraction.
EDTA: Divalent metal ion chelating agents such as Ca2+and Mg2+can inhibit the activity of DNase (divalent cation dependent) and disrupt the cell wall of Gram negative bacteria.
(2) Solution II: 0.2N NaOH; 1% SDS.
NaOH: plays a major role in cell lysis and can instantly dissolve the cell membrane, causing it to undergo a phase change from a bilayer membrane structure to a microcapsule structure. Although DNA generally only undergoes denaturation under alkaline conditions without hydrolysis of phosphodiester bonds, under such alkaline conditions, if the action time is too long, genomic DNA will still slowly break. Therefore, strict attention should be paid to the lysis time and the gentleness of solution mixing.
SDS (Anionic Detergent): Disrupts the non covalent bonds (hydrogen and hydrophobic bonds) of proteins and binds to the hydrophobic sites of proteins, causing denaturation, dissolution, and loss of natural conformation and function.
(3) Solution III: 3M potassium acetate; 2M acetic acid.
Precipitation: Protein precipitated by salt precipitation and acid denaturation; Precipitation occurring under low temperature and high salt concentration of SDS; Co precipitation of genomic DNA.
3M potassium acetate (KAc): SDS precipitates at high salt concentrations, making it easy for SDS to bind to proteins. High salt causes SDS/protein/polysaccharide complexes to precipitate; At the same time, K+ions increase the amount of precipitation (sodium dodecyl sulfate encounters potassium ions and becomes potassium dodecyl sulfate (PDS), which is insoluble).
Because bacterial genomic DNA is bound to proteins and is too long, long DNA is prone to aggregation and co precipitation by PDS, even though SDS does not bind to DNA molecules.
2M acetic acid: Neutralize NaOH because prolonged alkaline conditions can cause genomic DNA to break, so it needs to be neutralized. Once genomic DNA breaks, PDS cannot co precipitate fragments as long as they are 50-100kb in size; So the alkaline treatment time should be short and not violent oscillation, otherwise there will always be a large amount of genomic DNA mixed in on the final plasmid, and a relatively large genomic DNA band will be observed in agarose electrophoresis.
2) Phenol/chloroform/isoamyl alcohol (25/24/1) purification
Although the formation of PDS precipitation precipitates most of the proteins, there are still many proteins that cannot be precipitated, so purification treatment with phenol/chloroform/isoamyl alcohol is necessary.
Phenol: Organic solvent, which has a much greater denaturation effect on proteins than chloroform, but the proportion of water saturated phenol is slightly heavier than water. When it comes into high concentration salt solutions, the phenol phase will run to the aqueous layer after centrifugation, which is not conducive to the recovery of plasmid containing aqueous phase.
Chloroform: Adding chloroform can increase the specific gravity, keeping phenol/chloroform in the lower layer, facilitating the recovery of the aqueous phase; Phenols and water have great mutual solubility, and the residue of phenols can inhibit many enzyme reactions. Therefore, a mixture of phenols/chloroform should be used for extraction. The amount of phenols that run into the aqueous phase is much less, and trace amounts of phenols will be removed completely during ethanol precipitation.
Isopentanol: Its main function is to reduce the bubbles generated during protein denaturation, making the interface between the upper and lower layers clearer after centrifugation, and facilitating the recovery of the aqueous phase.
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3) Anhydrous ethanol precipitation
After purification with phenol/chloroform/isoamyl alcohol, a large amount of salt is retained in the water phase. As long as 2 times the volume of ethanol is added, the plasmid DNA can be precipitated by centrifugation after being left at room temperature for a few minutes.
Ethanol is highly soluble in water, taking away the water molecules around DNA. After DNA loses water, hydrogen bonds are easily formed between DNA molecules, making them prone to polymerization and precipitation.
In a solution with a pH of around 8, DNA molecules are negatively charged, and a large amount of salt ions (Na+/K+) are retained in the aqueous phase to neutralize the negative charge on DNA molecules, reducing the repulsive force between DNA molecules with the same charge phase. As a result, DNA sodium salts are easy to polymerize and precipitate with each other.
4) Electrophoretic identification
When identifying plasmid DNA by agarose gel electrophoresis, three bands can be seen in most cases, but not necessarily the three bands of super helix, linear and open loop. Because plasmid samples obtained by alkaline extraction generally do not contain linear DNA, EcoRI can be used to linearize the plasmid and then perform agarose electrophoresis. It can be seen that the position of linear plasmid DNA is different from the position of these three bands.
In fact, these three bands are sorted according to the speed of electrophoresis, namely super helix, open loop, and replication intermediate (i.e. two plasmids that have not been fully replicated are connected together). If you accidentally oscillate excessively after adding solution II, there will be a fourth band, which swims slowly and moves away from these three bands, resulting in fragments of 20-100kb E. coli genomic DNA. Coincidentally, sometimes the plasmids mentioned have 7-10 bands, which is due to the special DNA sequence leading to varying degrees of superhelix (the number of superhelixes varies).
2. Boiling method
The principle is that chromosomal DNA is much larger than plasmid DNA molecules, and there are linear molecules bound to chromosomal DNA, while plasmid DNA is a covalently closed circular molecule; When heating DNA solution, linear chromosome DNA is prone to denaturation, and covalently closed loop plasmid DNA returns to its natural conformation upon cooling; The denatured chromosome DNA fragments combine with denatured proteins and cell fragments to form precipitates, while the refolded supercoiled plasmid DNA molecules exist in a dissolved state in the liquid phase, which can be separated by centrifugation.
