Polynucleotide kinase, phosphorylation

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... 

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. 

OH groups. For ligation to restriction fragments 5 -phosphoryIated ends are required and the first step is the phosphorylation of these groups using polynucleotide kinase ... 

Restriction enzymes which cleave double-stranded DNA, making staggered cuts which leave the 5 -end of each strand extended. Double-stranded DNA with protruding 5 -ends is more efficiently phosphorylated by polynucleotide kinase than DNA with flush or recessed 5 -ends. The fore-shortened 3 -ends can also be labelled by extending them with 32P-labelled nucleoside triphosphates complementary to bases in the extended template strand using DNA polymerase. Y—pyrimidine nucleotide, R—purine nucleotide, N—any nucleotide. A complete list of commercially obtainable restriction endonucleases is given in Appendix III. 

An alternative to the two-stage end-labeling protocol involves a phosphate exchange reaction catalyzed by polynucleotide kinase. In the presence of excess amounts of ADP, polynucleotide kinase transfers a 5 -phosphoryl residue from DNA or RNA to ADP in a reversal of the phosphorylation reaction (5,12). At pH... 

An alternative method to prepare correct termini for the ligation with T7 RNA ligase is to dephosphorylate both ends of the 5 -fragment with E. coli alkaline phosphatase and phosphorylate both ends of the 3 -fragment with T4 polynucleotide kinase.71 72... 

Restriction-site mapping in DNA may be performed by end-labelling with polynucleotide kinase as above, and subsequent partial digestion with the restriction enzyme. An overlapping series of polynucleotides with a common labelled terminus is thus formed, and size analysis of these affords a restriction map. Polynucleotide kinase may also be used to assay the number of 5 -phosphorylated termini in DNA generated as the result of the action of restriction endonuclease. In the presence of... 

First, the linker is covalently joined to the ends of a DNA fragment or vector. For example, the 5 ends of a decameric linker and a DNA molecule are phosphorylated by polynucleotide kinase and then joined by the ligase from T4 phage (Figure 5.11). This ligase can form a covalent bond between blunt-ended (flush-ended) double-helical DNA molecules. 

For mutagenesis to introduce the stop codons, the protocols in Subheadings Oligonucleotide Phosphorylation with T4 Polynucleotide Kinase, Annealing of the Oligonucleotide to the Template, and step 1 of Enzymatic Synthesis of CCC-dsDNA can be scaled down by a factor of ten. The reaction product can be used directly to transform E. coli using any standard procedure. 

An alternative method to introducing, e.g., a polydeoxyadenylate tail onto DNA without the use of terminal deoxynucleotidyl transferase consists in 5 -phosphorylation of an appropriately tailed oligodeoxyribonucleotide, e.g. d(AATTCCCGGGAio), using T4 polynucleotide kinase, followed by annealing to d(CCCGGG) to create a hybrid containing an overlap end identical to that formed by restriction of DNA with Eco This hybrid is in turn annealed with Eco RI-... 

Novogrodsky, A. Tal, M. Traub, A. Hurwitz, J. The enzymatic phosphorylation of ribonucleic acid and deoxyribonucleic acid. II. Further properties of the 5-hydroxyl polynucleotide kinase. J. Biol. Chem., 241, 2933-2943 (1966)... 

Lillehaug, J.R. Kleppe, R.K. Kleppe, K. Phosphorylation of double-stranded DNAs by T4 polynucleotide kinase. Biochemistry, 15, 1858-1865 (1976)... 

Prinos, R Slack, C. Lasko, D.D. 5 Phosphorylation of DNA in mammalian cells identification of a polymin P-precipitable polynucleotide kinase. J. Cell. Biochem., 58, 115-131 (1995)... 

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