Biotechnology Index	Glossary

Polynucleotide Kinase

Polynucleotide kinase (PNK) is an enzyme that catalyzes the transfer of a phosphate from ATP to the 5' end of either DNA or RNA. It is a product of the T4 bacteriophage, and commercial preparations are usually products of the cloned phage gene expressed in E. coli. The enzymatic activity of PNK is utilized in two types of reactions: In the "forward reaction", PNK transfers the gamma phosphate from ATP to the 5' end of a polynucleotide (DNA or RNA). The target nucleotide is lacking a 5' phosphate either because it has been dephorphorylated or has been synthesized chemically. In the "exchange reaction", target DNA or RNA that has a 5' phosphate is incubated with an excess of ADP - in this setting, PNK will first transfer the phosphate from the nucleic acid onto an ADP, forming ATP and leaving a dephosphorylated target. PNK will then perform a forward reaction and transfer a phosphate from ATP onto the target nucleic acid. As you might expect, the efficiency of phosphorylating via the exchange reaction is considerably less than for the forward reaction. In addition to its phosphorylating activity, PNK also has 3' phosphatase activity, although this has little significance to molecular tecnologists. There are two major indications for phosphorylating nucleic acids and hence use of PNK are: Phosphorylating linkers and adaptors, or fragments of DNA as a prelude to ligation, which requires a 5' phosphate. This includes products of polymerase chain reaction, which are typically generated using non-phosphorylated primers. Radiolabeling oligonucleotides, usually with 32P, for use as hybridization probes. PNK is inhibited by small amounts of ammonium ions, so ammonium acetate should not be used to precipitate nucleic acids prior to phosphorylation. Low conceptrations of phosphate ions, or NaCl concentrations greater than about 50 mM, also inhibit this enzyme.

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Last updated on January 22, 2000

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