(a) The cutting of DNA at specific locations is possible with the help of ‘molecular scissors’ called restriction endonuclease. Restriction endonucleases make highly specific internal cuts in the DNA strand. These enzymes recognise palindromic sites within the DNA duplex and cut its strands. Their single stranded free ends are called sticky ends which can be joined end to end by DNA ligases.

 

(b) DNA fragments can be separated by gel electrophoresis. In this process, DNA strands (negatively charged) are separated by forcing them to move towards the anode under an electric field through a medium/matrix. Now-a-days the most commonly used matrix is agarose gel which is a natural polymer extracted from sea weeds. 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.

 

(c) In pBR322, selectable markers are antibiotic resistance gene that help to identify transformants and eliminate non-transformants. In case of pBR322, there are present two resistance genes tetracycline resistance (tetR) and ampicillin resistance (ampR).