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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 ↔ ADP + 5'-phospho-DNA
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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.
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Source:
T4 PNK is purified
from E. coli cells expressing a recombinant clone
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Common
reaction buffer:
The buffer contains 70 mM Tris-HCl, 10 mM MgCl2, 5 mM DTT and pH 7.6 at 25°C
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Structure:
Structure of the 5′‐kinase
domain. Two enzyme‐bound
sulfates coordinated at the active site tunnel are shown as a stick models.
Coordination of one sulfate within the oxyanion hole formed by the P‐loop is depicted by dashed lines.
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References
https://worldwide.promega.com/products/cloning-and-dna-markers/molecular-biology-enzymes-and-reagents/t4-polynucleotide-kinase/?catNum=M4101
By:
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Adrian Barnutiu A86084
·
Carlos Rodrigues A85152
·
Catarina Deseyve A85111
·
Gabriel Abreu A84397
·
Matilde Rocha A85740
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