Plasmid DNA
Preparation of plasmid DNA
Purification of plasmids from a culture of bacteria involves the same general strategy as preparation of total cell DNA.
A culture of cells, containing plasmids, is grown in liquid medium, harvested, and a cell extract prepared. The protein and RNA are removed, and the DNA probably concentrated by ethanol precipitation.
However, there is an important distinction between plasmid purification and preparation of total cell DNA. In a plasmid preparation it is always necessary to separate the plasmid DNA from the large amount of bacterial chromosomal DNA that is also present in the cells.Separating the two types of DNA can be very difficult, but is nonetheless essential if the plasmids are to be used as cloning vectors. The presence of the smallest amount of contaminating bacterial DNA in a gene cloning experiment may easily lead to undesirable results.
Fortunately several methods are available for removal of bacterial DNA during plasmid purification, and the use of these methods, individually or in combination, can result in isolation of very pure plasmid DNA.
The methods are based on the several physical differences between plasmid DNA and bacterial DNA, the most obvious of which is size.
The largest plasmids are only 8% of the size of the E. coli chromosome, and most are much smaller than this. Techniques that can separate small DNA molecules
from large ones should therefore effectively purify plasmid DNA.
In addition to size, plasmids and bacterial DNA differ in conformation.
When applied to a polymer such as DNA, the term conformation refers to the overall spatial configuration of the molecule, with the two simplest conformations
being linear and circular. Plasmids and the bacterial chromosome are circular, but during preparation of the cell extract the chromosome is always
broken to give linear fragments. A method for separating circular from linear molecules will therefore result in pure plasmids.
1) Separation on the basis of size
The usual stage at which size fractionation is performed is during preparation of the cell extract. If the cells are lysed under very carefully controlled conditions,
only a minimal amount of chromosomal DNA breakage occurs. The resulting DNA fragments are still very large - much larger than the plasmids and can be removed with the cell debris by centrifugation.
This process is aided by the fact that the bacterial chromosome is physically attached to the cell envelope, so fragments of the chromosome sediment with the cell debris if these attachments are not broken.
Cell disruption must therefore be carried out very gently to prevent wholesale breakage of the bacterial DNA. For E. coli and related species, controlled
lysis is performed. Treatment with EDTA and lysozyme is carried out in the presence of sucrose, which prevents the cells from bursting immediately. Instead, sphaeroplasts are formed, cells with partially degraded cell walls that retain an intact cytoplasmic membrane.
Cell lysis is now induced by adding a non-ionic detergent such as Triton X-lOO (ionic detergents, such as SDS, cause chromosomal breakage). This method causes very little breakage of the bacterial DNA, so centrifugation leaves a cleared lysate, consisting almost entirely of plasmid DNA.
A cleared lysate will, however, invariably retain some chromosomal DNA.
Furthermore, if the plasmids themselves are large molecules, they may also sediment with the cell debris. Size fractionation is therefore rarely sufficient on its own, and we must consider alternative ways of removing the bacterial DNA contaminants.
2) Separation on the basis of conformation
Before considering the ways in which conformational differences between plasmids and bacterial DNA can be used to separate the two types of DNA,
we must look more closely at the overall structure of plasmid DNA. It is not strictly correct to say that plasmids have a circular conformation, because
oouble-stranded DNA circles can take up one of two quite distinct configurations.
Most plasmids exist in the cell as supercoiled molecules. Supercoiling occurs because the double helix of the plasmid DNA is partially unwound during the plasmid replication process by enzymes called topoisomerases. The supercoiled conformation can be maintained only if both polynucleotide strands are intact, hence the more technical name of covalently closed-circular (ccc) DNA. If one of the polynucleotide strands is broken the double helix reverts to its normal relaxed state, and the plasmid takes on the alternative conformation, called open-circular(oc).
Supercoiling is important in plasmid preparation because supercoiled molecules can be fairly easily separated from non-supercoiled DNA.
Two different methods are commonly used. Both can purify plasmid DNA from crude cell extracts, although in practice best results are obtained if a cleared lysate is first prepared.
(i) Alkaline denaturation
The basis of this technique is that there is a narrow pH range at which nonsupercoiled DNA is denatured, whereas supercoiled plasmids are not.
If sodium hydroxide is added to a cell extract or cleared lysate, so that the pH is adjusted to 12.0-12.5, then the hydrogen bonding in non-supercoiled DNA
molecules is broken, causing the double helix to unwind and the two polynucleotide chains to separate.
If acid is now added, these denatured bacterial DNA strands reaggregate into a tangled mass. The insoluble network can be pelle ted by centrifugation, leaving plasmid DNA in the supernatant.
An additional advantage of this procedure is that, under some circumstances (specifically cell lysis by SDS and neutralization with sodium acetate), most of
the protein and RNA also becomes insoluble and can be removed by the centrifugation step. Further purification by organic extraction or column chromatography may therefore not be needed if the alkaline denaturation method is used.
(ii) Ethidium bromide-caesium chloride density gradient centrifugation
This is a specialized version of the more general technique of equilibrium or density gradient centrifugation. A density gradient is produced by centrifuging a solution of caesium chloride (CsCl) at a very high speed.
Macromolecules present in the CsCI solution when it is centrifuged form bands at distinct points in the gradient. Exactly where a particular molecule bands depends on its buoyant density; DNA has a buoyant density of about 1.7 g/cm3, and therefore migrates to the point in the gradient where the CsCI density is also 1.7 g/cm3. In contrast, protein molecules have much lower buoyant densities, and so float at the top of the tube, whereas RNAforms a pellet at the bottom.
Density gradient centrifugation can therefore separate DNA, RNA and protein and is an alternative to organic extraction or column chromatography for DNA purification.
More importantly, density gradient centrifugation in the presence of ethidium bromide (EtBr) can be used to separate supercoiled DNA from non-supercoiled molecules. Ethidium bromide binds to DNA molecules by intercalating between adjacent base pairs, causing partial unwinding of the double helix. This unwinding results in a decrease in the buoyant density, by as much as 0.125 g/cm3 for linear DNA. However, supercoiled DNA, with no free ends, has very little freedom to unwind, and can only bind a limited amount of EtBr. The decrease in buoyant density of a supercoiled molecule is therefore much less, only about 0.085 g/cm3. As a consequence, supercoiled molecules form a band in an EtBr-CsCl gradient at a different position to linear and open-circular DNA.
Ethidium bromide-caesium chloride density gradient centrifugation is a very efficient method for obtaining pure plasmid DNA. When a cleared lysate is subjected to this procedure, plasmids band at a distinct point, separated from the linear bacterial DNA, with the protein floating on the top of the gradientand RNA pelleted at the bottom. The position of the DNA bands can be seen by shining ultraviolet radiation on the tube, which causes the bound EtBr to fluoresce. The pure plasmid DNA is removed by puncturing the side of the tube and withdrawing a sample with a syringe. The EtBr bound to the plasmid DNA is extracted with n-butanol and the CsCl removed by dialysis. The resulting plasmid preparation is virtually 100% pure and ready for use as a cloning vector.
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Sources:
Content:
Gene Cloning and Analysis, an introduction(fifth edition), T. A. Brown
Images:
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