The following points highlight the three main steps involved in the extraction of DNA. The steps are: 1. Isolation of DNA 2. Purification of DNA 3. Removal of Unwanted RNA.
Extraction of DNA: Step # 1.
Isolation of DNA:
Initially, isolating DNA was a long and difficult process with large amounts of DNA collected. Extracting DNA from plants, animals, and bacteria, all require that the cellular contents be liberated into a solution. Since the bacteria are single-celled and contain no bone, fat, gristle, etc., the DNA is relatively easy to be extracted.
In contrast, samples from animals and plants must often be ground into tiny fragments before proceeding. Since plant cells have very rigid cell walls, the researcher must mechanically break the cells open in a blender, or add special degradative enzymes to digest the cell wall components.
Similarly, to extract DNA from a mouse’s tail, enzymes are added to degrade the connective tissue and disperse the cells. By far the easiest way to get DNA is to extract it from bacteria. A few drops of a bacterial culture will give plenty of DNA for most of the purposes. First, the bacterial cell wall is easily digested by lysozyme, an enzyme that degrades the peptidoglycan layer of the cell wall.
A successive treatment with detergent dissolves the lipids of the cell membrane. Chelating agents, such as EDTA (Ethylene Diamine Tetra acetate), are also used, especially with gram-negative bacteria, to remove the metal ions that bind components of the outer membrane together. In all these samples, the cellular contents, including the DNA, are then liberated into solution and are purified by further series of steps.
Extraction of DNA: Step # 2.
Purification of DNA:
Two general types of procedure are used for purification of DNA:
1. Centrifugation and
The principle of centrifugation is as follows:
The sample is spun at high speed and the centrifugal force causes the larger or heavier components to sediment at the bottom of the tube. For example, destroying the cell wall of bacteria by lysozyme and detergents leaves a solution containing fragments of the cell wall, which are small, and the DNA. When the sample is centrifuged, DNA and some other large components are sedimented at the bottom of tube.
The fragments of cell wall, together with many other soluble components, remain in solution and are discarded. The sedimented DNA is then re-dissolved in an appropriate buffer solution. Still it has a lot of protein and RNA mixed in with it.
These are generally removed by chemical means. One step used in many DNA purifications is phenol extraction. Phenol, also known as carbolic acid, is very corrosive and extremely dangerous because it dissolves and denatures the proteins that make up 60 to 70 per cent of all living matters. Consequently, phenol may be used to dissolve and remove all of the proteins from a sample of DNA.
When phenol is added to water, the two liquids do not mix to form a single solution; instead, the denser phenol forms a separate layer below the water. When shaken, the two layers mix temporarily, and proteins dissolve in phenol.
When the shaking stops, the DNA solution and phenol containing proteins separated into two layers (Fig. 3.1). To ensure that no phenol is trapped with the DNA, the sample is centrifuged briefly. Then the water containing DNA and RNA is sucked off and kept. Generally, several successive phenol extractions are performed to purify proteins from DNA.
A variety of newer techniques have been developed that avoid phenol extraction. Most of these involve purifying DNA by passing it through a column containing a resin that binds DNA but not other cell components.
The techniques in this regard are of following two types;
1. Affinity Chromatography:
This uses silica resins. Silica resins bind nucleic acids rapidly and specifically at low pH and high salt concentrations. The nucleic acids are released at higher pH and low salt concentration.
2. Ion-exchange Chromatography:
Anion exchange resins, such as diethyl amino-ethyl-cellulose, are positively charged and bind DNA via its negatively charged phosphate groups. In this case binding occurs at low salt concentrations and the nucleic acids are eluted by high concentrations of salt, which disrupt the ionic bonding.
Extraction of DNA: Step # 3.
Removal of Unwanted RNA:
Special enzymes remove contaminating RNA from a DNA sample. The enzyme ribbon clease degrades RNA into short oligonucleotides but leaves the giant DNA macromolecule unchanged. A mixture of DNA and RNA is first incubated with the ribonuclease at the optimal temperature for enzyme activity.
Next, an equal volume of alcohol is added. The alcohol precipitates large macromolecules, including long chains of DNA, out of solution. However, the small RNA fragments remain dissolved.
It should be noted that alcohol treatment is not very specific and will precipitate most large carbohydrates and many proteins as well as intact macromolecules of both DNA and RNA. Thus, alcohol precipitation can only be used after these components have been removed from the DNA by centrifugation and phenol extraction.
Next the DNA is sedimented at the bottom of the tube by centrifugation, and the supernatant solution containing the RNA fragments is discarded (Fig. 3.2). The tiny pellet of DNA left at the bottom of the tube is often scarcely visible. Nonetheless, it contains billions of DNA molecules, sufficient for most investigations.