T4 Polynucleotide Kinase

Detailní popis

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).