T4 Polynucleotide Kinase (PNK) catalyses the transfer of the γ-phosphate from ATP to the 5´ hydroxyl of ssDNA and dsDNA, RNA, and nucleoside 3´ monophosphates. The enzyme also removes the 3´ phosphate from 3´-phosphoryl polynucleotides, deoxyribonucleoside 3´ monophosphates, and deoxyribonucleoside-3´,5´-diphosphates to form a 3´-hydroxyl group.
Applications
Labeling of 5´ termini of DNA and RNA with 32P or 33P for DNA sequencing, blot-hybridization experiments, or transcript mapping using Mung Bean Nuclease, S1 nuclease, or other nucleases.1,2 Phosphorylation of oligonucleotide linkers and other DNA or RNA molecules prior to ligation, or for use in ligation amplification reactions with Ampligase® Thermostable DNA Ligase. Preparation of labelled DNA or RNA molecular weight markers for gel electrophoresis and chromatography.
Figure 1. The absence of DNA exonuclease activity in Epicentre's T4 Polynucleotide Kinase. 40-mer oligonucleotides with (5´-PO4) or without (5´-OH) phosphorylated 5´-termini were incubated with the indicated number of units of T4 PNK in 1X T4 PNK Reaction Buffer for 16 hours at 37°C. Electrophoresis of the incubation mixtures in a 15% polyacrylamide/8 M urea gel demonstrates no degradation of the oligonucleotides.
References
Sambrook, J. et al. (1989) in: Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, New York. Chaconas, G. et al. (1980) Meth. Enzymol. 65, 75. read more
Key features
Phosphorylate 5' hydroxy ends of ssDNA and dsDNA, RNA, and nucleoside 3 monophosphates
Highly active enzyme that readily phosphorylates 5' hydroxyl ends of nucleic acids
Flexible: Use newly phosphorylated nucleic acids in a variety of applications such as cloning, next gen sequencing library prep, and preparation of labelled nucleic acids
Product Details
Each T4 Polynucleotide Kinase Kit includes T4 Polynucleotide Kinase at 10 U/µL and 10X Reaction Buffer without ATP. ATP is available separately.
The "enzyme only" format contains T4 Polynucleotide Kinase at 10 U/µL.
