Introduction
Generally, Plasmid purification from the culture of bacterial cells has a similar strategy like preparation of total cell DNA. A cell culture having plasmids is grown in a medium to prepare cell extract. The RNA and different proteins are removed from the whole extract by using different techniques. After that, precipitation of DNA is being done by ethanol precipitation. But there is an important difference between the preparation of total cell DNA and purification of the plasmid. It is compulsory to separate the plasmids from the whole content of chromosomal DNA of a bacterial cell that also exists in the cell.
Basic principle of plasmid DNA isolation
It is very difficult to separate two types of DNA from each other but on the other hand it is necessary if these plasmids are to be used in cloning as a cloning vector. Even the presence of small amount of DNA can easily change the original results. Luckily, there are several methods that can be used to purify the plasmids. Basically, these processes do separation on the basis of physical differences between chromosomal DNA and plasmids like their size, which is the most obvious difference. The size of largest plasmid of E.coli is only 8% of the chromosomal DNA. Therefore, techniques that can separate out the large DNA molecules from the small sized DNA can be easily used to purify plasmids.
Furthermore, chromosomal DNA of bacteria and plasmids also differ with respect to their conformation it means they have different spatial conformations of the molecules. As plasmids have circular and chromosomal DNA has linear conformation, but due to breaking down of chromosomes during the process of extraction usually linear fragments are formed. Therefore, the processes that can separate the circular plasmids from the linear DNA fragments are being used.
Procedure of plasmid DNA isolation
There are two different bases on which plasmids are being separated from chromosomal DNA such as:
- Separation on the basis of size
- Separation on the basis of conformation
Separation of plasmid DNA on the basis of size
Larger fragments of chromosomal DNA are easier to separate out as compared to small broken fragments, so controlled disruption of the cell is needed. If cells are gently lysed under specific conditions then a small amount of chromosomal DNA will be a break. Therefore, DNA fragments are much larger with respect to plasmids and these broken fragments can be removed by centrifugation from the cell extract with cell debris. Naturally, chromosomal DNA fragments are also physically linked with the envelope of the cell, therefore, these attachments are not broken then chromosomal DNA fragments sediment along with the cell debris.
To minimize the break down of bacterial DNA during the process, cell disruption should be carried out very gently. So, controlled lysis is being done for E.coli and related species.
In the presence of sucrose, lysozyme and EDTA are used to treat the bacterial cells. This treatment protects the cells and prevents them from bursting instantly. Sucrose maintains the osmotic pressure which prevents the bursting of cell. EDTA donates electron pairs to the divalent metal cations and binds with them to form a chelate. These metal ions are cofactors for DNAses and necessary for their activity. Therefore, DNAses cannot degrade the DNA molecules. Lysozymes are enzymes that can catalyze the linkages of peptidoglycan in the cell wall of bacteria and help to remove it. Hence,sphaeroplasts are formed that are cells with an intact cytoplasmic membrane and have partially degraded cell wall around it. A non-ionic detergent such as Triton X-100 is used to lyse the plasma membrane gently. Ionic detergents are not suitable to rupture cell membrane they can cause break down of chromosomal DNA. These small pieces of DNA that have a size similar to plasmids may cause problems as they are difficult to isolate from the plasmid. Molecules of Triton X-100 has polar and nonpolar regions that may interact with the lipid bilayers and increase the permeability of the plasma membrane and eventually, total cell content comes out. Very little breakage of chromosomal DNA is caused by this method, hence, after centrifugation, a clear lysate is made containing plasmids.
However, the quantity of chromosomal DNA may differ in the cleared lysate. Moreover, sometimes plasmids also have large size and may sediment at the bottom with cell debris.
Separation of plasmid DNA on the basis of conformation
Plasmids and chromosomal DNA have many conformational differences between them. Plasmids do not have only circular conformation but also have many other conformations with respect to conditions. Mostly plasmids have supercoiled configuration in the cell also termed as covalently closed circular DNA. During replication of the plasmid DNA topoisomerases partially unwind it. This supercoiling of the plasmid DNA is only maintained if both polynucleotide strands remain intact. Many plasmids have relaxed state due to the damage of one polynucleotide strand of the DNA and then open circular conformation is attained.
(a) supercoiled—both strands are intact
(b) open-circular—one or both strands are nicked
Supercoiled molecules of plasmid DNA can be isolated more easily from non-supercoiled DNA molecules. Mostly following methods are used to isolate the plasmid DNA from crude cell extract.
1. Alkaline denaturation
The principle of the alkaline denaturation method is that there is a narrow range of pH that can denature the non-supercoiled DNA but has no effect on the supercoiled plasmids. If pH 12.0-12.5 is maintained in the cell extract for a while by adding sodium hydroxide, then hydrogen bonding in two polynucleotide chains of non-supercoiled DNA is broken. Due to this double helix of that non-supercoiled DNA will unwind. After that acid is being added to the cell extract as a result denatured strands of non-supercoiled DNA reaggregate and make tangled mass. By centrifugation, the reaggregated tangle mass can be pelleted out. Plasmid DNA goes into the supernatant layer by centrifugation. Further purifications are not needed after alkaline denaturation.
2. Ethidium bromide-caesium chloride density gradient centrifugation
This is a specific version of density gradient centrifugation. The density gradient is made by high-speed centrifugation of caesium chloride (CsCl). There are macromolecules in the CsCl that make different bands at distinct points. The buoyant density of DNA is almost 1.70 g/cm3, therefore, DNA molecules go to the point where the density of CsCl is 1.70 g/cm3. RNA molecules settle down in the form of the pallet, whereas proteins may float at the surface because of lower buoyant densities. After that, different methods are used to separate DNA from proteins and RNA, like column chromatography and organic extraction for DNA purification. At present, the density gradient centrifugation is mostly used.
(a) A CsCl density gradient produced by high
speed centrifugation. (b) Separation of protein,
DNA, and RNA in a density gradient.
In the presence of ethidium bromide (EtBr), density gradient is being used to separate non-supercoiled DNA from supercoiled DNA molecules. Ethidium bromide binds and intercalates between adjacent base pairs of DNA molecules and partially unwinds the double helix. Therefore, the buoyant density of linear DNA strand decreases by 0.125 g/cm3. But, the decrease in the density of supercoiled DNA is much less by 0.085 g/cm3 due to the absence of free ends and little freedom of unwinding. As a result, open circular or linear and supercoiled DNA molecules form their bands at different sites in the EtBr-CsCl gradient.
normal DNA molecule shown on the left is partially unwound by taking up four EtBr molecules, resulting in the “stretched” structure on the right.
By Ethidium bromide-cesium chloride density gradient method, pure plasmids can be obtained easily by subjecting cleared lysate to this procedure. As plasmids make their band at a different point with respect to linear bacterial DNA and RNA molecules make their pallet at the bottom and proteins keep floating at the top of the gradient. By throwing ultraviolet radiations, DNA bands can be seen due to the fluorescence caused by bounded EtBr. By puncturing the side of the tube, pure plasmid DNA can be extracted by using a syringe. CsCl is removed by the process of dialysis and the plasmid bounded EtBr is extracted by using n-butanol the resulting vector is ready to use for the cloning purposes.
