Structural Biochemistry/T4 Polynucleotide Kinase

Description

The T4 Polynucleotide Kinase (T4 PNK) catalyzes the transfer of the phosphate from ATP to the 5'-OH group of single- and double-stranded DNAs and RNAs as well as oligonucleotides. The reaction can be reversed. The enzyme is also a 3'-phosphatase and a homotetramer. It consists of four identical subunits of 28.9 kDa. The T4 polynucleotide kinase can be used in a buffer for restriction enzymes in techniques such as PCR. It can be found in E. coli with a duplicated gene of the bacteriophage T4. It can also be used for labeling 5’-terminus of nucleic acids which can be used as probes for hybridization, transcript mapping; markers for gel electrophoresis. They can also be used as primers for DNA sequencing and PCR technique as well as an enzyme that removes the 3’-phosphate groups.

Storage

T4 Polynucleotide Kinase should be stored in -20°C

Applications

The T4 polynucleotide kinase is used for labeling the 5'-termini of nucleic acids, and the labeled products can be used in the following ways: - markers for gel-electrophoresis - primers for DNA sequencing - primers for PCR - probes for hybridization - substrates for DNA and RNA ligases - probes for transcrip mapping 5'-phosphorylation of oligonucleotide linkers and DNA or RNA is required before ligation can be performed. using the ³²P-postlabeling assay, DNA modification can be detected.

Miscellaneous

The 5'-termini of nucleic acids can be labeled either through the forward or the exchange reaction. When the forward reaction is carried out, the phosphate from [³²P or ³³P]-ATP is transferred to the 5'-hydroxyl end of DNA or RNA. If the nucleic acid already contains a 5'phosphate, though, that portion is removed either through the use of calf intestine alkaline phosphatase or bacterial alkaline phosphatase. When the exchange reaction is carried out, the nucleic acid 5'-phosphate is transferred to ADP and the radiolabeled phosphate from [³²P or ³³P]-ATP is then transferred to the nucleic acid that does not contain a 5'-hydroxyl group. The ideal buffer to use for the exchange reaction is Imidazole-HCl buffer, which has a pH of 6.4. PEG is used to improve the rate and efficiency of the kinase reaction and should be added to the exchange reaction. When phosphate and ammonium ions are present, the T4 polynucleotide kinase is inhibited. Prior to phosphorylation reaction, DNA should not be precipitate if ammonium ions are present.