T4 Polynucleotide Kinase

Available On-Site

B buffer for 100% activity FastDigest buffer for 100% activity G buffer for 100% activity O buffer for 100% activity Recombinant enzyme R buffer for 100% activity Tango buffer for 100% activity Thermal inactivation at 75°C in 10 min

T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the gamma-phosphate from ATP to the 5'-OH group of oligonucleotides, ss and dsDNA and RNA.
  
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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).

Highlights

  • Active in Thermo Scientific restriction enzyme, RT, and T4 DNA Ligase buffers

Applications

  • Labeling of nucleic acids' 5'-termini(see​ Reference3, 4) to be used as (see Figure 1 in Supporting Data):
    • probes for hybridization
    • probes for transcript mapping
    • markers for gel electrophoresis
    • primers for DNA sequencing
    • primers for PCR
  • 5'-phosphorylation of oligonucleotide, PCR products, other DNA or RNA prior to ligation
  • Phosphorylation of PCR primers
  • Detection of DNA modification by the [32P]-postlabeling assay(see​ Reference5, 6)
  • Removal of 3'-phosphate groups(see​ Reference2)

Notes

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).

  
10X Reaction Buffer A (for forward reaction)500mM Tris-HCl (pH7.6 at 25°C), 100mM MgCl2, 50mM DTT, 1mM spermidine
10X Reaction Buffer B (for exchange reaction)500mM imidazole-HCl (pH6.4 at 25°C), 180mM MgCl2, 50mM DTT, 1mM spermidine and 1mM ADP
24% PEG Solution24%(w/v) polyethylene glycol 6000
Concentration10 U/µL
Definition of Activity Unit
  • One unit of the enzyme transfers 1nmol of gamma-phosphate from ATP to 5'-OH DNA in 30min at 37°C.
  • Enzyme activity is assayed in the following mixture: 100mM Tris-HCl (pH8.0), 10mM MgCl2, 5mM DTT, 0.5mM 5'-OH DNA, 0.05mM ATP and 0.1MBq/mL [gamma-33P]-ATP.
HazardousNo
InactivationInactivated by heating at 75°C for 10min or by addition of EDTA.
InhibitionInhibitors: metal chelators, phosphate and ammonium ions, KCl and NaCl at a concentration higher than 50mM
Molecular WeightThe enzyme is a homotetramer. It consists of four identical subunits of 28.9kDa.
Quality ControlThe absence of endo-, exodeoxyribonucleases and ribonucleases confirmed by appropriate quality tests. Functionally tested for labeling 5'-termini of DNA.
Storage BufferThe enzyme is supplied in 20mM Tris-HCl (pH7.5), 25mM KCl, 0.1mM EDTA, 2mM DTT and 50%(v/v) glycerol.
Storage Condition-20 C
Labeling efficiency of DNA containing different types of 5'-ends

Labeling efficiency of DNA containing different types of 5'-ends

Labeling efficiency of DNA containing different types of 5'-ends

Labeling efficiency of DNA containing different types of 5'-ends.


References

  1. 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).
  2. C. C. Richardson, Bacteriophage T4 polynucleotide kinase, The Enzymes (Boyer, P.D., Ed., Academic Press, San Diego, 14, 1981) pp. 299-314.
  3. J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual, the Third edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).
  4. F. M. Ausubel et al., Current Protocols in Molecular Biology, vol. 1 (John Wiley, New York, 2001).
  5. D. H. Phillips, Detection of DNA modifications by the 32P-postlabelling assay.Mutation Res.378, 1-12 (1997).
  6. G. Keith, G. Dirheimer, Postlabeling: a sensitive method for studying DNA adducts and their role in carcinogenesis.Curr. Opin. Biotechnol.6, 3-11 (1995).
  7. B. Harrison, S. B. Zimmerman, T4 polynucleotide kinase: macromolecular crowding increases the efficiency of reaction at DNA termini.Anal. Biochem.158, 307-315 (1986).

Citations