Question

Alkaline phosphatase is used to

• A. Add the phosphate group onto free 5' terminus of a DNA molecule
• B. Add one or more deoxyribonucleotides onto the 5' terminus of a DNA molecule
• C. Remove the phosphate group at 5' terminus of a DNA molecule
• D. Change the conformation of covalently closed circular DNA

Hint:

Alkaline phosphatase removes 5'-phosphate groups from linearized plasmid vectors.
This prevents vector self-ligation, increasing the efficiency of foreign DNA insert ligation during cloning.

Answer 1

The Correct Option is C

Step 1: Understanding the Concept:
Alkaline phosphatase is an enzyme that catalyzes the removal of phosphate groups from the 5'-terminal ends of nucleic acid molecules (DNA and RNA).
This enzymatic dephosphorylation is an important tool in molecular cloning to prevent unwanted self-ligation of linearized vector plasmids.

Step 2: Detailed Explanation:
During cloning, plasmid vectors are linearized using restriction endonucleases.
This cleavage leaves a 5'-phosphate group and a 3'-hydroxyl group at both ends of the DNA molecule.
In the presence of DNA ligase, these cohesive or blunt ends can readily rejoin, causing the vector to recircularize without incorporating the target insert DNA.
To prevent this, the linearized vector is treated with alkaline phosphatase (such as Calf Intestinal Alkaline Phosphatase, CIAP, or Shrimp Alkaline Phosphatase, SAP).
The enzyme catalyzes the hydrolytic removal of the 5'-phosphate groups, leaving 5'-hydroxyl ends.
Since DNA ligase requires a 5'-phosphate and a 3'-hydroxyl to form a phosphodiester bond, dephosphorylated vector ends cannot self-ligate.
Ligation can only occur when the target insert DNA (which retains its 5'-phosphate groups) is introduced, ensuring the successful formation of recombinant plasmids.

Step 3: Final Answer:
Alkaline phosphatase is used to remove the phosphate group at the 5' terminus of a DNA molecule, which corresponds to Option (C).