Kinase 353
- If the oligonucleotide is not labeled highly enough, add another 10 U of
enzyme and incubate for a further 30 min. - Purify as required.
Note: Equal concentrations of ATP and 5' ends gives 50% labeling.
To obtain high specific activity, increase the ATP:oligonucleotide ratio
to 10:1. Only 10% of the label will be transferred, but virtually every
oligonucleotide molecule will be labeled and to a specific activity
approaching that of the ATP (27). - Summary
Polynucleotide kinase provides a simple means of modifying the
termini of nucleic acids. It facilitates the labeling of molecules, or the
preparation of those molecules for further manipulation. It can be used
in concert with other enzymes such as alkaline phosphatase and DNA
ligase to enable a series of manipulations to be carried out, or it can
perform useful functions alone. The enzyme is especially important
for the modification of short synthetic oligonucleotides, but its ability
to act on a variety of substrates makes it a tool with a wide range of
applications.
References- Spencer, E., Loring, D., Hurwitz, J., and Monroy, G. (1978) Enzymatic con-
version of 5'-phosphate terminated RNA to 5'-di- and triphosphate terminated
RNA. Proc. Natl. Acad. Sci. USA 75, 4793-4797 - Richardson, C. C. (1965) Phosphorylation of nucleic acid by an enzyme from
T4 bacteriophage infected Escherichia coli. Proc. Natl. Acad. Sci. USA 54,
158-163 - Novogrodsky, A., Tal, M., Traub, A., and Hurwitz, J. (1966) The enzymatic
phosphorylation of ribonucleic acid and dexoxyribonucleic acid. II. Further
properties of the 5'-hydroxyl polynucleotide kinase. J. Biol. Chem. 241,
2933-2943. - Ichimura, M. and Tsukada, K. (1971) Polynucleotide kinase from rat liver
nuclear extracts. J. Biochem. 69, 823-828. - Austin, G. E., Sirakoff, D., Roop, B., and Moyer, G. H. (1978) Purification
and properties of polynucleotide kinase of calf thymus. Biochim. Biophys. Acta
522, 412-422. - Zimmerman, S. B. and Pheiffer, B. H. (1981) Eukaryotic Kinases, in The
Enzymes, 3rd ed., vol. 14 (Boyer, P. D., ed.), Academic, New York, pp.
315-329. - Shuman, S. and Hurwitz, J. (1979) 5'-hydroxyl polyribonucleotide kinase from
HeLa cell nuclei. Purification and properties. J. Biol. Chem. 254, 10,396-10,404.