Practice question

Alkaline phosphatase hydrolyzes 5'-phosphomonoester bonds, converting 5'-phosphate termini of linearized plasmid DNA into 5'-hydroxyl groups. DNA ligase strictly requires a 5'-phosphate and adjacent 3'-hydroxyl to catalyze phosphodiester bond formation, so dephosphorylated vector molecules cannot self-circularize, thereby suppressing empty vector background. In cloning, the foreign insert retains chemically intact 5'-phosphates supplied by restriction digestion or polynucleotide kinase, enabling ligation at each junction to form a heteroduplex with two nicks that are repaired after transformation by bacterial repair machinery, greatly improving recombinant recovery.