Botany · Ch 4 — Principles and Processes of Biotechnology
Alkaline Phosphatase
Alkaline Phosphatase
Alkaline phosphatase solves a specific, easily overlooked problem in vector preparation. When a circular vector like a plasmid is cut open by a restriction enzyme, both of its newly exposed ends still carry a 5'-phosphate group - which means DNA ligase can, and very often will, simply reseal the vector back into its original closed circle before any foreign DNA insert gets the chance to join in (self-ligation, also called recircularisation). Alkaline phosphatase prevents this by removing that terminal 5'-phosphate group from the cut vector ends. Without a 5'-phosphate on either end, DNA ligase can no longer catalyse a bond there, so the cut, dephosphorylated vector simply cannot reseal itself - it can only be joined to a piece of DNA (the foreign insert) that still has its own intact phosphate group, which is exactly what forces the ligation reaction to favour producing genuine recombinants rather than empty, self-recircularised vector. …
What this figure shows. Two parallel reaction panels contrasting outcomes for a linear plasmid cut open to expose two ends bearing free phosphate (P) and hydroxyl (OH) groups. Top panel: with alkaline phosphatase absent, DNA ligase directly rejoins the two ends of the same cut plasmid, recircularising it by self-ligation with no foreign insert (an unwanted outcome). Bottom panel: alkaline phosphatase first removes the terminal phosphate groups (releasing 2 Pi) from the cut plasmid ends, after which DNA ligase can no longer reseal the plasmid on its own ('No reaction' / X marked over that path) - so the vector stays open and can only be sealed once a foreign DNA fragment supplies the missing phos …