Q3. Write various application of shrimp alkaline phosphatase enzyme in molecular cloning experiments.

Ans: Shrimp alkaline phosphatase (SAP) enzyme removes 5' phosphate group from DNA, RNA, dNTPs and proteins. Various application of SAP are-

i. Dephosphorylate 5'-phosphate group of DNA/RNA for subsequent labeling of the ends.

ii. To prevent self-ligation of the linearized plasmid.

iii. To prepare PCR product for sequencing.

iv.  To inactivate remaining dNTPs from PCR product (for downstream sequencing appication).

Q4. What are the types of reactions catalyzed by PNK enzyme?

Ans: PNK carries out two types of enzymatic activity-

i.  Forward reaction: PNK can convert 3' PO₄ /5' OH ends into 3' PO₄ /5' PO₄ ends which blocks further ligation by ligase enzyme. γ-phosphate of ATP is transferred to the 5' end of a polynucleotide (DNA or RNA). 5' phosphate is not present either due to chemical synthesis or dephosphorylation. Asp35 of PNK forms the co-ordinate bond with 5' OH and attacks γ phosphorus forming an intermediate.

 ii. Exchange reaction: PNK also catalyses the reverse reaction by targeting DNA or RNA having a 5' phosphate. DNA/RNA is incubated with an excess of ADP and PNK transfers the phosphate from the nucleic acid to an ADP, forming ATP. PNK then performs a forward reaction and transfer a phosphate from ATP to the target nucleic acid. Exchange reaction is used to label with radioactive phosphate group.