• Shrimp alkaline phosphatase (SAP) - Shrimp alkaline phosphatase is highly specific, heat labile phosphatase enzyme isolated from arctic shrimp ( Pandalus borealis ). It removes 5' phosphate group from DNA, RNA, dNTPs and proteins. SAP has similar specificity as CIP but unlike CIP, it can be irreversibly inactivated by heat treatment at 65°C for 15mins. SAP is used for 5' dephosphorylation during cloning experiments for various application as follows:
► Dephosphorylate 5'-phosphate group of DNA/RNA for subsequent labeling of the ends.
► To prevent self-ligation of the linearized plasmid.
► To prepare PCR product for sequencing.
► To inactivate remaining dNTPs from PCR product (for downstream sequencing appication).
Two primary uses for alkaline phosphatase in DNA modification:
• Removing 5' phosphate from different vector like plasmid, bacteriophage after treating with restriction enzyme. This treatment prevents self ligation because unavailability of phosphate group at end. So, this treatment greatly enhances the ligation of desired insert. During ligation of desired insert, the complementary ends of the insert and vector will come to proximity of each other (only for sticky ends but not for blunt ends). One strand of the insert having 5'-phosphate will ligate with the 3'OH of the vector and the remaining strand will have a nick. This nick will be sealed in the next step by ligase enzyme in the presence of ATP. It is used to remove 5' phosphate from fragment of DNA prior to labeling with radioactive phosphate.
2-3.2 Methylase:
• Methyltransferase or methylase catalyzes the transfer of methyl group (-CH3) to its substrate. The process of transfer of methyl group to its substrate is called methylation.
• Methylation is a common phenomenon in DNA and protein structure.
• Methyltransferase uses a reactive methyl group that is bound to sulfur in S-adenosyl methionine (SAM) which acts as the methyl donor.
• Methylation normally occurs on cytosine (C) residue in DNA sequence. In protein, methylation occurs on nitrogen atom either on N-terminus or on the side chain of protein.
• DNA methylation regulates gene or silence gene without changing DNA sequences, as a part of epigenetic regulation.
• In bacterial system, methylation plays a major role in preventing their genome from degradation by restriction enzymes. It is a part of restriction – modification system in bacteria.