Catalyses the transfer of the γ-phosphate from ATP to the 5'-hydroxyl group of single- and double-stranded DNAs and RNAs, oligonucleotides, or nucleoside 3'-monophosphates (forward reaction).

• Detection of DNA modification by the [³²P]-postlabelling assay
• 5'-phosphorylation of oligonucleotides, PCR products, and DNA or RNA prior to ligation
• Removal of 3'-phosphoryl groups
• Phosphorylation of PCR primers

T4 polynucleotide kinase exhibits 5'-phosphatase activity and catalyses the exchange of phosphate groups between 5'-P-oligo-polynucleotides and ATP (exchange reaction). The enzyme is also a 3'-phosphatase.

Labelling 5'-termini of nucleic acids to be used as probes for hybridisation, probes for transcript mapping, markers for gel electrophoresis, primers for DNA sequencing, primers for PCR.