CAS Number: 37211-65-7
MDL No.: MFCD00166009
Storage: -20C
Enzyme Commission (EC) Number: 2.7.1.78 ( BRENDA | IUBMB )
UNSPSC Code: 12352204
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 muM 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 30Â min. at 37Â C. Transfer is detected as incorporation into acid-insoluble material.
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 [gamma-32P]-ATP.
RIDADR: NONH for all modes of transport
WGK Germany: 3