Question

Unless the vector and source DNA are cut, fragments separated and joined, the desired recombinant vector molecule cannot be created.
(a) How are the desirable DNA sequences cut ?
(b) Explain the technique used to separate the cut fragments.
(c) How are the resultant fragments joined to the vector DNA molecule ?

Answer 1

(a) The desirable DNA sequences are cut using the restriction end nucleases enzymes which cut the DNA strand a little away from the centre of the palindromic sites.
(b) The cut fragments can be separated using gel electrophoresis. In this technique the DNA fragments are forced to move towards the anode under an electric field through the agarose matrix. The separated DNA fragments are visualized after staining with a compound such as ethiduim bromide followed by exposure to UV radiation.
(c) The resultant fragments are joined to the vector DNA molecule using the enzyme DNA ligase.