Cold Spring Harb Protoc. 2020 Aug 3;2020(8):100669. doi: 10.1101/pdb.prot100669.
The removal of 5' phosphates from nucleic acids is used to enhance subsequent labeling with [γ-P]-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 -nitrophenyl phosphate (PNPP) and the exposed 5' phosphates of nucleic acids to bulky globular protein substrates.
核酸 5' 磷酸基团的去除用于增强随后用 [γ-P]-ATP 的标记，减少连接反应中质粒载体的环化，并使 DNA 易受或抵抗作用于核酸的其他酶（例如 λ 核酸外切酶）的影响。本质上，任何核苷酸磷酸酶（例如细菌碱性磷酸酶、小牛肠碱性磷酸酶 [CIP]、胎盘碱性磷酸酶、虾碱性磷酸酶 [SAP] 或几种酸性磷酸酶，如甘薯和前列腺酸性磷酸酶）都将催化核酸模板 5' 磷酸基团的去除。事实上，这些酶更喜欢小分子底物，如 -硝基苯膦酸酯 (PNPP) 和核酸的暴露 5' 磷酸基团，而不是大的球状蛋白底物。
