Kinase polynucleotide

Polynucleotide kinase j Transfers terminal phosphate (y position) i from ATP to 5 -OH groups of DNA or RNA. P labeling of DNA or RNA. 

Cameron, V., Soltis, D. and Uhlenbeck, O. C. Polynucleotide kinase from a T4 mutant which lacks the 3 phosphatase activity . Nucleic Acids Res., 5,825-833 (1978). 

Many other enzymes can be used to remove or add groups to the ends of the DNA molecule. The three major ones are alkaline phosphatase, which removes a phosphate group from the 5 terminus polynucleotide kinase, which adds a phosphate group to a free 5 terminus and terminal deoxynucleotidyl transferase, which adds one or more deoxynucleotides to the 3 terminus. 

In this method the single-stranded DNA fragment to be sequenced is end-labelled by treatment with alkaline phosphatase to remove the 5 phosphate, followed by reaction with 32P-labelled ATP in the presence of polynucleotide kinase, which attaches 32P to the 5 terminal. The labelled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base as follows. 

Separation and Quantitation.—The specific radioactivity of [y-32P]ATP of very high activity (up to 240 Ci per millimole) may be measured by using it to phosphorylate [dT(pT)10] quantitatively, using polynucleotide kinase, isolating the labelled undecanucleotide, and measuring its activity.170 Isotope dilution is used to confirm the values. An alternative method of measuring specific radioactivities of ribo-nucleoside triphosphates utilizes a 3H-labelled nucleoside triphosphate (e.g. UTP) of unknown specific activity, a 14C-labelled nucleoside (e.g. ATP), a suitable primer in... 

Kits and enzymes Superscript reverse transcriptase (Invitrogen), Maxiscript and Megascript in vitro transcription kits (Ambion, Austin, TX). Taq-DNA polymerase, T4-polynucleotide kinase, EcoRI and Hindlll restriction enzymes (Invitrogen). 

Treat the purified DNA fragment by T4 polynucleotide kinase at 37°C for 1 h, then purify the DNA fragment by ethanol precipitation. 

DNA polymerase I (E. coli) Reverse transcriptase Polynucleotide kinase Terminal transferase Exonuclease III... 

Another important procedure is labeling ends of polynucleotides. Most often the 5 end is labeled with a radioisotope or by covalent attachment of a fluorescent dye. For example, a polynucleotide kinase can be used to transfer a radioactive y-phospho group from ATP to the 5 end of a polynucleotide that has a free 5 -OH group. 

T4 polynucleotide kinase Bacteriophage T4 Phosphorylation of 5 -OH terminus of a polynucleotide (DNA or RNA)... 

Koch et a1.1401 developed a method in which a peptide sequence (H-Leu-Arg-Arg-Ala-Ser-Leu-Gly-OH, the Kemptide ) was synthesized either at the N-terminal or the C-terminal residue of the PNA. This peptide is a substrate for protein kinase A, which phos-phorylates the critical Ser residue. This phosphorylation was carried out using [y-32P]ATP. Kozlov et al. 45 made 2 -deoxycytidine-3 -phosphoroimidazolide to react with the N-terminus of a PNA in solution. T4 polynucleotide kinase then phosphorylated the 5 -hydroxy group of the nucleotide by [y-32P]ATP. 

Fig. 4. Scheme of the preparation containing P-labeled phosphomonoesters at single-strand breaks. The two strands of Ti DNA duplex are schematically represented by two parallel lines and only the 5 termini are designated. After the introduction of single-strand breaks into DNA by incubation with pancreatic DNase, the phosphomonoesters formed are removed by phosphatase at 65°. The 5 termini are then labeled by incubation with polynucleotide kinase. From Weiss el al. (56). 

Lorsch, J. R., and J. W. Szostak, In vitro evolution of new ribozymes with polynucleotide kinase activity. Nature 371 31-36, 1994. 

Weinfeld M, Liuzzi M, Paterson MC (1990) Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites design of a 32P-postlabeling assay for apurinic sites in DNA. Biochemistry 29 1737-1743... 

The pEU3b plasmid was digested with Spe I and blunted with T4 polynucleotide kinase. The plasmid was then digested with Sal I. The coding region for yeast ubiquitin was prepared as follows. 

Small ribozymes and DNAzymes leave 2 —3 cyclic phosphate at the site of cleavage, and this group must be removed before the ligation. This task can be accomplished by treating cleaved products with polynucleotide kinase (PNK) in the absence of ATP (Schurer et al., 2002). 

P-Radiolabeled RNA substrate to be cleaved (prepared by reaction of the RNA with y-32P-ATP and T4 polynucleotide kinase alternatively, 3/-32P-radiolabeling with 32P-pCp and T4 RNA ligase can be used)... 

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