Kinase 349
End-labeling allows the quantification of the termini, the enzyme
often being used in conjunction with alkaline phosphatase to assess
the number of 5'-phosphate groups present by measuring the available
5'-hydroxyl groups before and after phosphatasing. It can also be used
to characterize cleavage points in nucleic acids.
End-labeled oligonucleotides can be used as primers for sequencing
(33). Both oligonucleotides and polynucleotides can be used as hybrid-
ization probes (34-36) for clone or genomic characterization, restric-
tion mapping using partial digestion techniques, DNA or RNA
fingerprinting (37,38), DNA footprinting (39,40), nuclease S 1 analy-
sis, physical mapping, and sequence analysis (41).
Phosphorylation reactions where the phosphate moiety is unlabeled
(or labeled purely for the purposes of monitoring the reaction) are used
in the synthesis of substrates for DNA or RNA ligation (42). These
may be vector molecules, genomic fragments, or synthetic oligonucle-
otides such as linkers. Such manipulations allow the assembly of long
nucleic acid molecules from short synthetic precursors.
The mutant 3'-phosphatase-free enzyme is especially useful for RNA
analysis since the continued presence of the 3'-phosphate group pre-
vents cyclization of concatenation (23).
Finally the wild-type enzyme may be used as a specific Y-phos-
phatase under the right conditions (43).
4.2. Storage and Stability
Enzyme preparations are commercially available at concentrations
of 1000-12,000 U/mL, and with specific activities in the range 30,000-
40,000 U/mg.
The enzyme is normally stable for up to 18 mo at-20°C in a suitable
storage buffer. The composition of this buffer can vary, but a general
purpose buffer used for the storage of a wide variety of enzymes can
be used, with the optional addition of 0.1-1.0 jxM ATP. An example of
a typical storage buffer would be: 50 mM Tris-HCl, pH 7.5; 25 mM
KCI; 1 mM DTT; 0.1 mM EDTA; 0.1 ~/ATP; and 50% glycerol.
4.3. Reaction Conditions
T4 polynucleotide kinase will act in restriction enzyme buffers allow-
ing simultaneous restriction digestions (31), but specific conditions
have also been defined as described in Section 3.9. Reaction condi-