Isolation and Purification of DNA (Bacteria, Plant and Animal Cell)

The basic procedure requires releasing DNA from cells and purifying DNA for use in experiments.

DNA isolation and purification mainly includes four steps:

Culture of bacterial or eukaryotic cells (freshly prepared).
The cells that need to be lysed to release their components.
For bacterial cell lysis, chemical required lysozyme, with EDTA and a detergent such as SDS.
EDTA removes divalent cations and therefore destabilizes the outer membrane in bacteria such as E. coli and also inhibits DNA which otherwise tends to degrade DNA, while detergents solubilize lipids in the membrane.
Plant and fungal cells require alternative treatments, both mechanical and enzymatic (cellulase, chitinase).
Animal cells (without a cell wall) can generally be lysed by a softer treatment with a gentle treatment . detergents (such as CTAB).
Complex solution of DNA, RNA, proteins, lipids and carbohydrates.
Sudden lysis of the cell generally causes chromosomal DNA fragmentation.
Bacterial plasmids are easily obtained in their native circular state using standard lysis conditions.
The next step in the procedure is to separate the desired nucleic acid from other components.

The removal of RNA from a DNA preparation by treatment with ribonuclease (RNase A).
RNase is a very heat stable enzyme, it is easy to make sure that it is free of traces of deoxyribonucleases (DNases) which would otherwise degrade DNA, simply by heating the enzyme before using it.
Protein contamination can be removed by digestion with a proteolytic enzyme such as proteinase K (optional step).

Phenol and chloroform are immiscible with water, get two layers (phases) once added to the cell extract.
When the mixture is vigorously stirred, the proteins denature and precipitate at the interface.
If using phenol balanced with a neutral or alkaline buffer, the nucleic acids (DNA and RNA) will remain in the aqueous layer.
On the other hand, if acid phenol extraction is performed, the DNA divides into the organic phase, allowing RNA to be recovered from the aqueous phase.
Phenol is naturally acidic, so balancing it with water or using an acidic buffer will produce the appropriate conditions.
Phenol extraction is also useful in the later stages of handling when it is necessary to make sure that all traces of enzymes have been removed before proceeding to the next step.

Adding an alcohol, or isoporpanol or more frequently ethanol; in the presence of monovalent cations (Na +, K + or NH4+) a nucleic acid precipitated forms which can be collected on the bottom of the test tube by centrifugation.
Part of the salt will precipitate at will and will be removed by washing with 70% ethanol.

Removal of cellular debris or the recovery of precipitated nucleic acids. It is also frequently used in various column purification methods.
In addition to these methods, an ancient method of separating nucleic acids, or any type of macromolecule, is to subject them to ultracentrifugation in a density gradient.
This included the use of the cesium salt gradient, generally with the addition of ethidium bromide, for the separation of plasmid DNA from bacterial genomic DNA or DNA/RNA.
In addition, the sucrose gradient can be used for selecting the size of large DNA fragments when building a genomic library.
DNA is separated due to size and / or configuration difference (i.e., supercoiled versus relaxed).

Extraction and purification procedure for plasmid DNA, bacterial cells with the desired plasmid are lysed (decomposed) under alkaline conditions and the crude lysate (cell debris) is purified by filters or centrifugation.
The lysate is then loaded into an apparatus in which plasmid DNA binds selectively under appropriate conditions of low salt and pH content.
RNA, proteins, metabolites and other low molecular weight impurities are removed with a medium salt wash, then the plasmid DNA is released into a high salt buffer.
DNA can be concentrated and desalted for genetic engineering uses.
In the bacterial cell extract, chromosomal DNA exists as linear fragments, due to the fragmentation that occurs during the lysis of the cell.
Raising the pH to about 12 will disrupt the hydrogen bonds and allow the linear supports to separate.
Plasmids are more resistant and are not affected by cell lysis; they remain as intact coiled circular DNA.
Although the pH will alter the hydrogen bonds, the two circular chains cannot be physically separated and will be interconnected.
When the pH is lowered, the interconnected plasmid supports will reconnect to reform the double-stranded plasmid.
On the other hand, separate linear chromosomal fragments cannot do this; instead they will be added to an insoluble net that can be removed during centrifugation.

3.5 Column purification:

Two types of column purification are used, with the help of centrifugation (spin column).
In size selection chromatography, a sample is passed through a series of small porous beads.
Smaller molecules, such as salts and unincorporated nucleotides, will enter the pearls, while larger molecules, such as longer nucleic acid, will pass through the column.
This type of purification is a valuable tool and often a simple and quick alternative to the purification of alcoholic precipitations.
In affinity chromatographic purification, the macromolecules in the sample will bind to the resins in the column.
This could be an anionic resin that binds to groups of phosphates negatively charged in the nucleic acid skeleton or more sophisticated, such as resins coated with oligo-dT sequences that bind specifically to the poly-A tail of eukaryotic mRNA molecules.
In both cases, undesirable molecules can be washed from the column, after which stringent conditions are changed and the bound nucleic acids are eluted into a small water or buffer.

Estimate the DNA concentration by measuring the absorbance of the solution in a spectrophotometer at 260 nm.
This is convenient, but not very sensitive; a 50 µg / ml solution of ds DNA will have an absorbance of 1.
However, the presence of proteins and phenol will influence this estimate.
Furthermore, UV absorbance does not allow you to verify the integrity of your DNA; it could be completely degraded and still give a reading.
Accurate quantification can be performed by performing DNA or RNA on gel electrophoresis.
Protein or RNA contamination can be estimated by UV absorbance at 260 and 280 nm.
If the ratio 260/280 is high, it indicates RNA contamination and if the ratio is low, it means protein contamination.
Dyes such as EtBr are commonly used to detect and quantify nucleic acids.
EtBr has a flat ring structure that can be stacked between nucleic acid bases; this is known as intercalation.
The dye can be detected by its fluorescence when exposed to UV rays.
This is the most widely used method for coloring electrophoresis gels and can also be used to estimate the amount of DNA or RNA in the sample by comparing the intensity of the fluorescence with a sample of known concentration in the same gel.

Source: Microbiology notes

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