Dephosphorylation of DNA Fragments with Alkaline Phosphatase

Abstract

The removal of 5′ phosphates from nucleic acids is used to enhance subsequent labeling with [γ-³²P]-ATP, reduce the circularization of plasmid vectors in ligation reactions, and render DNA susceptible or resistant to other enzymes that act on nucleic acids (e.g., λ exonuclease). Essentially, any nucleotide phosphatase (e.g., bacterial alkaline phosphatase, calf intestinal alkaline phosphatase [CIP], placental alkaline phosphatase, shrimp alkaline phosphatase [SAP], or several acid phosphatases such as sweet potato and prostate acid phosphatase) will catalyze the removal of 5′ phosphates from nucleic acid templates. In fact, these enzymes prefer small substrates such as p-nitrophenyl phosphate (PNPP) and the exposed 5′ phosphates of nucleic acids to bulky globular protein substrates.