Practice question

DNA ligase catalyzes phosphodiester bond formation only between adjacent 3' hydroxyl and 5' phosphate termini. Vector linearized with single restriction enzyme retains complementary sticky ends bearing 5' phosphates, readily self-ligating and producing high background of empty clones. Alkaline phosphatase, including calf intestinal or shrimp isoforms, hydrolyzes 5' phosphate groups leaving 5' hydroxyl termini, rendering the vector ligation-incompetent unless paired with phosphorylated insert DNA supplying phosphates. Subsequent ligation yields circular recombinant product with one nick at each junction, later repaired after transformation into host bacteria.