Analysis Note

Activity is determined in a reaction mixture containing 40 mM Tris-HCl (pH 7.5), with 10 mM MgCl2, 5 mM dithiothreitol, 0.5 mM 5’;-OH polynucleotide ends, and mM [γ-32P]-ATP.

Application

Suitable for: Sequencing or nucleic acid tagging (DNA and RNA) by 5′-end labeling 5′ phosphorylation of oligonucleotides Removal of 3′-phosphate groups from phosphorylpolynucleotides

Components

T4 Polynucleotide Kinase is supplied in a solution of 50% glycerol (v/v), 20 mM Tris-HCl (pH 7.5), 25 mM KCl, 2mM DTT, 0.1 mM EDTA, and 0.1 µM ATP.

Principle

Polynucleotide kinase catalyses a 'forward reaction' transfer of the γ-phosphate of ATP to the 5′ hydroxyl terminus of single- and double-stranded nucleic acids (DNA and RNA) and 3′-nucleoside monophosphates. In exchange reactions containing ADP, the enzyme will catalyze the exchange of 5′-terminal phosphate groups and ATP. The 3′-phosphatase activity enables the enzyme to remove 3′-phosphoryl groups from phosphorylpolynucleotides. 1. Forward reaction: Transfer of the labeled γ-phosphate from [γ-³²P]-ATP to the free 5′-hydroxyl group of the substrate. 5′-HO-DNA + [γ-³²P]-ATP → 5′-³²PO-DNA + ADP.Substrates that do not have a free 5′-hydroxyl require prior dephosphorylation by alkaline phosphatase.2. Exchange reaction: First, the terminal 5′-phosphate is transferred from the substrate to ADP present in the reaction mixture. Then, the labeled γ-phosphate from [γ-³²P]-ATP is transferred to the free hydroxyl group of the substrate.5′-PO-DNA + ADP → 5′-HO-DNA + ATP5′-HO-DNA + [γ-³²P]-ATP → 5′-³²PO-DNA + ADP

Unit Definition

One unit catalyzes the transfer of one nanomole of ³²P to the 5′-end of micrococcal nuclease-treated DNA in 30 min. at 37 °C. Transfer is detected as incorporation into acid-insoluble material.