Previously added items:
Thermo Scientific T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the gamma-phosphate from ATP to the 5'-OH group of single- and double-stranded DNAs and RNAs, oligonucleotides or nucleoside 3'-monophosphates (forward reaction). The reaction is reversible. In the presence of ADP T4 Polynucleotide Kinase exhibits 5'-phosphatase activity and catalyzes the exchange of phosphate groups between 5'-P-oligo-polynucleotides and ATP (exchange reaction) (see Reference1).
The enzyme is also a 3'-phosphatase (see Reference2).
Polyethylene glycol (PEG) and spermidine improve the rate and efficiency of the phosphorylation reaction (see Reference7). PEG is used in the exchange reaction mixture.
As T4 Polynucleotide Kinase is inhibited by ammonium ions, use sodium acetate to precipitate DNA prior to phosphorylation(see References 1, 2).
Labeling efficiency of DNA containing different types of 5'-ends.
K. L. Berkner, W. R. Folk, Polynucleotide kinase exchange reaction: quantitave assay for restriction endonuclease-generated 5'-phosphoroyl termini in DNA.J. Biol. Chem.252, 3176-3184 (1977).
C. C. Richardson, Bacteriophage T4 polynucleotide kinase, The Enzymes (Boyer, P.D., Ed., Academic Press, San Diego, 14, 1981) pp. 299-314.
J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual, the Third edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).
F. M. Ausubel et al., Current Protocols in Molecular Biology, vol. 1 (John Wiley, New York, 2001).
D. H. Phillips, Detection of DNA modifications by the 32P-postlabelling assay.Mutation Res.378, 1-12 (1997).
G. Keith, G. Dirheimer, Postlabeling: a sensitive method for studying DNA adducts and their role in carcinogenesis.Curr. Opin. Biotechnol.6, 3-11 (1995).
B. Harrison, S. B. Zimmerman, T4 polynucleotide kinase: macromolecular crowding increases the efficiency of reaction at DNA termini.Anal. Biochem.158, 307-315 (1986).