Question

The extraction of DNA is a common procedure in biotechnology research laboratories. Research the methods of extracting DNA in biotechnology facilities. Describe how the extraction methods used in this lesson compare to those of research laboratories.

Answer 1

DNA extraction refers to the isolation of nucleic acid (DNA) through the disruption of cell membrane and nuclear membrane. The general principal of the extraction procedure involves the lysis of nuclear and cell membrane through mechanical (mortar-pestle), chemicals (detergents) or through enzymes (peptidase) and after this DNA extraction takes place thrrough various methods:

Chemical DNA extraction method:

It is a solution based DNA extraction procedure which can be divided up the use of either organic or inorganic solvents to extract the DNA.

Organic solvent includes Phenol Chloroform extraction: In this method the SDS (Sodium dodecyl sulfate) which is a detergent is used to the lyse the cell membrane and to prevent the hydrolysis of DNA through endogenous nucleases, this enzyme is chelated using Mg2++ ions using EDTA. Phenol and chloroform are used to denature and separate proteins from DNA and for the stabilization of the phases (aqueous and phenol). The denatured proteins form a layer at the interface between the aqueous and the organic phases which are removed by centrifugation. The DNA is then precipitated using ethanol.

Inorganic DNA extraction:

Proteinase K: To the extraction buffer which is common for all the extraction methods containing Tris, EDTA and SDS, this time an enzyme is added for the isolation of the DNA. Proteinase K enzyme is utilized for the digestion of protein and now can be used for the separation of the DNA. The DNA is prescipitated in alcohol and the sample is again centrifuged to remove all the cell debris. Finally, the DNA pellet is dissolved in TE buffer. This method is high yielding and rapid but the quality of the DNA is doubtful.

Solid phase extraction: (SIlica column based method)

Various DNA extraction kits are available nowadays which involve the DNA extraction through silica in which the DNA molecules bind and can be easily purified. This method is easy and rapid and no precipitation step is required.

As silica is positively charged molecules so it binds to the negatively charged DNA. After the addition of extraction buffer to the sample proteinase K digest protein and then the sample is centrifuged to remove all other impurities. Then with the silica column inserted in the microfuge tube the DNA binds to the silica. Then washing is done to improve the purity in which the aqueous phase containing the impurities are discarded. And finally the DNA bound to silica is eluted through TE buffer.

Physical extraction of DNA:

Magnetic beads:

In this method positively charged magnetic beads are used to attract the negatively charged DNA and it is extracted under the magnetic field.