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KAI-OLAF NETZER
• In an exchange reaction, the substrate is phosphorylated DNA. An excess
of ADP, however, first leads to dephosphorylation ofthe DNA which is
then rephosphorylated by transfer of 32 P y-phosphate.
T4 polynucleotide kinase (forward reaction)
The following gives a standard protocol for kinase mediated end-labeling of
a DNA oligonucleotide primer. The amount of 50 pmole labeled primer
would be sufficient to perform e.g. 50 direct, PCR mediated sequencing reactions (4 reactions [A, G, C, T] each).
Materials
Supplies
• Heating block (37°C)
• Alternatively: Thermal cycler
Reagents
• Dephosphorylated primer (1-50 pmol in approx. 1-201-11 water); the solution should be free from unincorporated nucleotides and low molecular
weight DNA which may decrease the efficiency of the T4 reaction; in addition, care should be taken that ammonium ions which strongly inhibit
T4 polynucleotide kinase are absent from the primer solution.
Primer length
ng of primer/pmol
17
5.6
19
6.3
21
6.9
23
7.6
25
8.2
27
8.9
29
9.6
• 10x buffer (500 mM Tris-HCl pH 7.6, 100 mM MgClz, 50 mM DTT, 1 mM
spermidine)
• y- 32 P ATP (>3000 Ci/mmol, 10!-!Ci/!-11, 3.3 1-1M)