Polynucleotide kinase labelling

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. 

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... 

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. 

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). 

Fig. 2.11. In (a), 3 -end labelling is achieved using deoxynucleotidyl transferase and an ff-[32P] ribonucleotide triphosphate followed by elimination of the ribonucleotide residues. In (b), 5 -end labelling is carried out using polynucleotide kinase and y-[32P]ATP. Partial digestion with spleen phosphodiesterase removes nucleotides sequentially from the 5 -end of the oligonucleotide giving a mixed population of partially degraded molecules each labelled at the 3 -end. Venom phosphodiesterase removes nucleotides from the 3 -end similarly yielding a mixed population of shortened fragments. The products of the reaction are resolved by two-dimensional electrophoresis-homochromatography and the sequence deduced by the characteristic...

Procedure D is a method for 5 -end labelling of a set of fragments, not previously dephosphorylated, using the polynucleotide kinase catalysed exchange reaction. 

Labelling 5 -ends with polynucleotide kinase and [y-i2P]-rATP (i) For protruding 5 -ends... 

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. 

The protocol for EMSA as recently noted by Xu et al. 2004 is as follows. Oligonucleotides are labeled using T4 polynucleotide kinase and [y -32P] ATP. The labeled probes are purified from free nucleotides by ethanol precipitation. 5 pL of extracted proteins is mixed with 2 xL of 5 x gel shift binding buffer and incubating at room temp for 15 min. Then 1 pL of 32P-labeled oligonucleotide is added and 20 min is given for a binding reaction to occur at room temp. 

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... 

Dephosphorylated oligonucleotides Polynucleotide kinase + AT p 5 -terminal P-labelled oligonucleotides Separation... 

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