BIOLOGY 3 Marks Question

→ Nucleic acid is the genetic material of all organisms without exception.
→ In majority of organisms this is deoxyribonucleic acid or DNA.
→ In order to cut the DNA with restriction enzymes, it needs to be in pure form, free from other macro-molecules.
→ Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids.
→ This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).
→ Genes are located on long molecules of DNA interwined with proteins such as histones.
→ The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease.
→ Purified DNA ultimately precipitates out after the addition of chilled ethanol.
→ This can be seen as collection of fine threads in the suspension.
→ DNA that separates out can be removed by spooling.

→ The techniques used for separation and isolation of DNA fragments is known as gel electrophoresis.
→ In this method Agarose gel is used as a medium.
→ The cutting of DNA by restriction endonucleases results in the fragments of DNA.
→ These fragments can be separated by a technique as shown in figure.

→ Since, DNA fragments are negatively charged molecules they can be separated by forcing them to move towards the anode under an electric field througha medium/matrix.
→ The DNA fragments separate (resolve) according to their size through sieving effect provided by the agarose gel.
→ Hence, the smaller the fragment size, the farther it moves.
→ The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation.
→ DNA appears as bright orange coloured bands of DNA in an ethidium bromide stained gel exposed to UV light.
→ The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution.
→ The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

→ Selection of recombinants due to inactivation of antibiotics is a cumbersome procedure because it requires simultaneous plating on two plates having different antibiotics.
→ Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate.
→ In this, a recombinant DNA is inserted within the coding sequence of an enzyme, ẞ-galactosidase. This results into inactivation of the gene for synthesis of this enzyme, which is referred to as insertional inactivation.
→ The presence of a chromogenic substrate gives blue coloured colonies if the plasmid in the bacteria does not have an insert.
→ Presence of insert results into insertional inactivation of the ẞ-galactosidase gene and the colonies do not produce any colour, these are identified as recombinant colonies.