CBSE Class 12-science Answered

Isolation of Genetic Material (DNA)  

• The DNA must be pure and free from other macromolecules such as RNA, proteins, polysaccharides and lipids for the action of restriction enzymes.  
• Pure DNA is obtained by treating the bacterial cell or plant and animal tissue with enzymes.
•  The enzyme lysozyme is used for digesting the bacterial cell wall, cellulose for the plant cell wall and  chitinase for the fungal cell wall.  
• RNA can be removed by treating the cells with ribonuclease, while the proteins can be removed by  treatment with protease.  
• The purified DNA finally precipitates out after the addition of chilled ethanol.
• DNA appears in the form of fine threads in the suspension.

Cutting of DNA at Specific Locations

• Purified DNA is cut by restriction enzyme under optimal conditions. 
• The restriction enzyme digest is run in agarose gel electrophoresis. 
• DNA is a negatively charged molecule, and so, it moves towards the positively charged electrode, the anode.
• The same procedure is then repeated with the vector DNA.
• After cutting the source DNA and the vector DNA with a specific restriction enzyme, the gene of interest from the source DNA and the cut vector are mixed, and the enzyme ligase is added to it.
• The ligase forms new sugar-phosphate bonds to join two DNAs.
• This results in the formation of rDNA or hybrid DNA or chimeric DNA.

Amplification of Gene of Interest using PCR

• Polymerase chain reaction (PCR) is a technique used to synthesise multiple copies of the desired gene in vitro within a short span of time.
• Amplification of a gene sample of interest is carried out using PCR in the following three steps:denaturation, annealing and extension.