T4 Polynucleotide Kinase

• Phosphorylation of 5ʹ ends of DNA prior to ligation

T4 Polynucleotide Kinase (PNK) catalyzes the transfer and exchange of the terminal gamma position phosphate of ATP to the 5ʹ-hydroxyl terminus of double- and single-stranded DNA, RNA and nucleoside 3ʹ -monophosphate molecules (1). T4 PNK also exhibits 3ʹ-phosphatase and 2ʹ, 3ʹ cyclicphosphodiesterase activities (2, 3, 4, 5, 6).

This enzme is supplied in 10 mM Tris-HCl, 50 mM KCl, 0.1 µM ATP, 1.0 mM DTT, 0.1 mM EDTA and 50% glycerol; pH 7.4 at 25°C.

10X Polynucleotide Kinase Buffer (cat. no. B9040) contains 700 mM Tris-HCl, 100 mM MgCl₂ and 50 mM DTT; pH 7.6 at 25°C.

SDS available upon request.

Enzyme properties

• Storage temperature: –25°C to –15°C
• Molecular Weight 34,620 Daltons

Source of recombinant enzyme protein
The protein is produced by a strain ofE. coli that expresses the recombinant T4 Polynucleotide Kinase gene.

Unit definition: One unit is defined as the amount of enzyme catalyzing the incorporation of 1 nmol of [32P] (ATP donor) in 30 minutes at 37°C in 1X T4 Polynucleotide Kinase Reaction Buffer.

References

1. Richardson, C.C. (1981) P.D. Boyer (Eds.), The Enzymes, 14, pp. 229-314. San Diego: Academic press.
2. Morse, D.P., et al. (1997) Biochemistry 36:8429.
3. Cameron, V. et al. (1977) Biochemistry 16:5120.
4. Wand, L.K. et al. (2002) Nucl. Acids Res. 30:1073.
5. Galburt, E., et al. (2002) Structure 10:1249.
6. Wang, L.K., et al. (2002) EMBO J. 21:3873

Quality control analysis

Unit activity was measured using a twofold serial dilution method. Dilutions of enzyme were made in 1X reaction buffer and added to 50 µl reactions containing 10 µM Oligo dT single-stranded DNA, 1X PNK Reaction Buffer, 66 µM ATP and [γ-32P] ATP. Reactions were incubated 30 minutes at 37°C, placed on ice, and analyzed using the method of Sambrook and Russell (Molecular Cloning, v3, 2001, pp. A8.256).

Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band corresponding to the protein of interest in the diluted sample.

Single-stranded exonuclease is determined in a 50 µl reaction containing a radiolabeled single-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded exonuclease activity is determined in a 50 µl reaction containing a radiolabeled double-stranded DNA substrate and 10 µl of enzyme solution incubated for 4 hours at 37°C.

Double-stranded endonuclease activity is determined in a 50 µl reaction containing 0.5 µg of plasmid DNA and 10 µl of enzyme solution incubated for 4 hours at 37°C.

This OEM by QIAGEN product is available for bulk purchase for the following commercial assay applications.

• DNA or RNA end labeling for probes and DNA sequencing
• Removal of 3ʹ-phosphate groups