| Product |
T4
Polynucleotide Kinase - MBI |
| Description |
Purified from an E.coli strain carrying
a plasmid with the cloned gene in coding this enzyme.
The enzyme catalyzes the transfer of the phosphate from
ATP to 5'-OH group of DNA, RNA, Oligonucleotides or nucleoside
3'-monophosphates. The reaction is reversible. T4 Polynucleotide
Kinase is also a 3'-phosphatase. It catalyzes the hydrolysis
of 3'-phosphoryl groups of deoxynucleoside 3'-monophosphates,
deoxynuleoside 3', 5'-diphosphatase and of 3'-phosphorylpolynucleotides. |
| Storage |
-20°C |
| Applications |
labeling 5'-termini of
nucleic acids, labeled products can be used as:
- Markers for gel-electrophoresis,
- Primers for DNA sequencing,
- Primers for PCR,
- Probes for hybridization,
- Substrates for DNA and RNA ligases,
- Probes for transcript mapping.
5'-phosphorylation of oligonucleotide linkers and DNA
or RNA prior to ligation.
Detection of DNA modification by the 32P-postlabeling
assay. |
| Note |
5'-termini of nucleic
acids can be labeled by either the forward or the exchange
reaction. In the forward reaction the phosphate from [32P
or 33P]-ATP is transfered to the 5'-hydroxyl end of DNA
or RNA. If the nucleic acid already contains a 5'-phosphate,
it is removed, using either calf intestine alkaline phosphatase
or bacterial alkaline phosphatase. In the exchange reaction,
the nucleic acid 5'-phosphate is transferred to ADP and
the radiolabeled phosphate from [32P or 33P]-ATP is subsequently
transferred to the nucleic acid free 5'-hydroxyl group.
The optimal buffer for the exchange reaction is Imidazole-HCl
buffer, pH 6.4.
· Polyethylene glycol (PEG)
improves the rate and efficiency of the kinase reaction.
PEG should be added to the exchange reaction.
·
T4 polynucleotide kinase is inhibited by phosphate and
ammonium ions. The DNA should not be precipitated in the
presence of ammonium ions prior to the phosphorylation
reaction. |
|
