Question

166. In which of the following scenario one cannot
distinguish between recombinant and
non-recombinant cell?
(1) When foreign DNA is inserted at Sall
recognition site in PBR322
(2) In PBR322, a foreign DNA is inserted at Hind III
recognition site
(3) When recombinant PBR322 was created by
inserting foreign DNA at Pvul recognition site
(4) Recombinant PBR322 which was introduced in
cell carries foreign DNA at Pst I recognition site​

Answer 1

Answer:

the answer is 2

Explanation:

(2) In PBR322, a foreign DNA is inserted at Hind III

recognition site

Answer 2

In PBR322, foreign DNA is inserted at Hind III recognition site - In this case, recombinants cannot be distinguished from non-recombinant cells (Option - 2)

• According to the structure of PBR322, all other insertion sites lie either in the ampicillin resistance region or in the tetracycline resistance region such as Pst I and Pvu I lie in the Ampicillin resistance region whereas SalI lies in the tetracycline resistance region.
• Checking if the insert has been added at the desired site is checked via 2 steps in Cloning.
• 1st the transformant is distinguished from non-transformant by checking resistance to the drug - in which the insert has not been inserted.
• Those that show resistance to the particular drug are the transformants.
• Recombinants are distinguished from non-recombinants by checking the resistance of previously selected transformants to the 2nd drug in which the gene has been inserted.
• The resistance to the drug in which the gene has been inserted will be lost due to gene disruption and recombinants will not survive in such conditions.
• Therefore, the gene must be inserted in one of the antibiotic resistance regions in order to distinguish between recombinant and non-recombinant cells.
• But Hind III recognition site lies in a region that does not disrupt any of the Antibiotic resistance genes, Hence, in this case, one cannot distinguish between recombinant and non-recombinant cells.