T4 Polynucloetide Kinase
catalyzes the transfer of the ¥ã-phosphate of ATP to a 5'-terminus of
polynucleotides (double-stranded and single-stranded DNA or RNA) and
nucleoside 3'-monophosphates. In the forward reaction, the ¥ã-phosphate is
transferred to the 5'-terminus of dephosphorylated DNA. In the exchange
reaction, an excess of ADP causes T4 polynucleotide kinase to transfer the
terminal 5'-phosphate from phosphorylated DNA to ADP, the DNA is then
rephosphorylated by transfer of a radiolabeled ¥ã-phosphate from
[¥ã-32P]ATP. This enzyme also contains a 3'-phosphatase
Recombinant strain of E. coli.
¡Ü 5' end labeling of
single- or double-stranded DNA and RNA for probes and for DNA sequencing.
¡Ü Addition of 5'-phosphates to synthetic linkers or DNA fragments that
lack terminal 5'-phosphates for subsequent ligation
One unit is defined as
the amount of enzyme required to catalyze the transfer of 1 nmol phosphate
from [¥ã-32P]ATP to the 5'-OH terminus of an oligonucleotide in 30 minutes
STANDARD UNIT ASSAY CONDITIONS
Tris-HCl (pH7.6), 10mM MgCl2, 5mM DTT, 66 ¥ìM [¥ã-32P]ATP, 0.26mM
5'-hydroxyl terminated salmon sperm DNA.
CONCENTRATION: 5-10 units/¥ìl.
10X REACTION BUFFER
(pH7.6@37¡É), 100mM MgCl2, 50mM DTT. Incubate at 37¡É.
10mM Tris-HCl (pH
7.4), 50mM KCl, 0.1mM EDTA, 1mM DTT, 0.1 ¥ìM ATP and 50% glycerol. Store at
1. ATP should be present at a
concentration of at least 1¥ìM in the forward reaction and at least 2¥ìM in
the exchange reaction, but optimal activity occurs at higher
2. T4 polynucleotide kinase is strongly inhibited by
ammonium ions. Therefore DNA should not be dissolved in, or precipitated
from, buffers containing ammonium ions prior to phosphorylation.
1. Richardson, C. C. (1981)
In The Enzymes (Boyer, P. D., ed.) Vol. 14, pp 299-314, Academic Press.
2. Maxam, A. M. and Gilbert, W. (1980) Methods Enzymol. 65, 499.
3. Richardson, C. C. (1972) Nucleic Acids Res. 2, 815.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A
Laboratory Manual, second ed., Cold Spring Harbor Laboratory, Cold Spring