Friday, November 23, 2018

T4 Polynucleotide Kinase



Ø  Definition:
T4 Polynucleotide Kinase catalyzes the transfer of a phosphate from ATP to the 5´-terminus of polynucleotides or to mononucleotides bearing a 5´-hydroxyl group. The enzyme may be used to phosphorylate RNA, DNA and synthetic oligonucleotides prior to subsequent manipulations such as ligation and cloning.
ATP + 5'-dephospho-DNA {\displaystyle \rightleftharpoons } ADP + 5'-phospho-DNA        

Ø  Applications:
The T4 polynucleotide kinase is used for labeling the 5'-termini of nucleic acids, and the labeled products can be used int he 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'-phosphorilation of oligonucleotide linkers and DNA or RNA is required before ligation can be performed. using the 32P-postlabeling assay, DNA modification can be detected.

Ø  Source:
 T4 PNK is purified from E. coli cells expressing a recombinant clone

Ø  Common reaction buffer:
The buffer contains 70 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT and pH 7.6 at 25°C

Ø  Structure:



















Structure of the 5′kinase domain. Two enzymebound sulfates coordinated at the active site tunnel are shown as a stick models. Coordination of one sulfate within the oxyanion hole formed by the Ploop is depicted by dashed lines.

Ø  References
https://worldwide.promega.com/products/cloning-and-dna-markers/molecular-biology-enzymes-and-reagents/t4-polynucleotide-kinase/?catNum=M4101

By:
·       Adrian Barnutiu A86084
·       Carlos Rodrigues A85152
·       Catarina Deseyve A85111
·       Gabriel Abreu A84397
·       Matilde Rocha A85740

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