Dephosphorylated DNA. 5 HO OH 3 + [γ- P]ATP 3 HO OH 5. FIGURE 1. T4 Polynucleotide Kinase Forward Reaction. OH 3 + ADP + [γ- P 5

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1 FOCUS Introduction ON APPLICATIONS T4 Polynucleotide Kinase Technical Bulletin T4 polynucleotide kinase contains a 5 kinase activity and a 3 phosphatase activity (1-3). It catalyzes the addition of the γ-phosphate of ATP to the 5 hydroxyl termini of DNA or RNA, including oligonucleotides. T4 polynucleotide kinase is used to add phosphate groups to 5 hydroxyl termini of linkers prior to ligation reactions (4, 5) or to add P-label to the 5 end of DNA (6) or RNA. End-labeled DNA is used for Maxam-Gilbert sequencing (7), as radiolabeled DNA markers, as hybridization probes (8-10), in S1 analysis of mrna (11, 12), and in mutagenesis protocols (13). T4 polynucleotide kinase can phosphorylate nucleic acids by either the forward or the exchange reaction. The forward reaction catalyzes the transfer of a terminal γ-phosphate of ATP to the 5 hydroxyl end of DNA or RNA (1, 2) (figure 1). If the DNA or RNA already contains a 5 phosphate group, this group must be removed to leave a 5 hydroxyl terminus before the addition of T4 polynucleotide kinase (14). In the exchange reaction, T4 polynucleotide kinase transfers the 5 phosphate group from DNA to ADP and subsequently transfers the γ-phosphate of ATP to the free 5 hydroxyl group to rephosphorylate the DNA (15) (figure 2). Since RNA typically does not have a 5 monophosphate, it is not a useful substrate for the exchange reaction. This is an equilibrium reaction forced to completion by a high concentration of ADP. Because the 5 phosphate group has been removed, the forward reaction is much Dephosphorylated DNA 5 HO OH 3 + [γ- P]ATP 3 HO OH 5 FIGURE 1. T4 Polynucleotide Kinase Forward Reaction. 5 P 3 HO OH 3 + ADP + [γ- P]ATP P 5 T4 Polynucleotide Kinase FIGURE 2. T4 Polynucleotide Kinase Exchange Reaction. more efficient than the exchange reaction. If labeled DNA of high specific activity is required, the forward reaction is the protocol of choice. Although it is less efficient, the exchange reaction is the easier protocol when using phosphorylated DNA because it requires fewer manipulations. GIBCO BRL T4 Polynucleotide Kinase is isolated from a recombinant E. coli strain overexpressing the T4 polynucleotide kinase gene. It is a tetramer composed of four identical subunits of 38 kda. One unit, as defined by Richardson (1, 2), incorporates 1 nmol of phosphate from [γ- P]ATP into acid-precipitable material in 30 min at 5 P 3 HO T4 Polynucleotide Kinase 5 P 3 HO OH 3 P 5 OH 3 + ATP + ADP P 5 37 C using micrococcal nuclease-treated DNA. T4 polynucleotide kinase is stored in 50 mm Tris-HCl (ph 7.6), 25 mm KCl, 5 mm DTT, 0.1 µm ATP, 0.2 mg/ml BSA, and 50% (v/v) glycerol. It has no detectable contaminating activity in ribonuclease, endodeoxyribonuclease, 3 and 5 exodeoxyribonuclease, and 5 phosphatase assays. The levels of incorporation into DNA fragments are determined in exchange and forward end labeling reactions. This bulletin describes conditions and considerations for labeling the 5 end of DNA or RNA using T4 polynucleotide kinase. TM

2 2 Materials In addition to T4 polynucleotide kinase and DNA or RNA, the following reagents and equipment may be necessary, depending on the protocol followed: Forward reaction buffer (5X); [350 mm Tris-HCl (ph 7.6), 500 mm KCl, 50 mm MgCl 2, 5 mm 2-mercaptoethanol]. Store at 20 C. This buffer is supplied with the GIBCO BRL T4 Polynucleotide Kinase. Exchange reaction buffer (5X); [250 mm Imidazole-HCl (ph 6.4), 60 mm MgCl 2, 0.35 mm ADP, 5 mm 2-mercaptoethanol]. Store at 20 C. This buffer is supplied with the GIBCO BRL T4 Polynucleotide Kinase. [γ- P]ATP ( 3,000 Ci/mmol) (10 mci/ml) 3 mm ATP 10 mm Tris-HCl (ph 7.5), 0.1 mm Na 2 EDTA (TE) 0.5 M Na 2 HPO M ammonium acetate 3.0 M sodium acetate 0.5 M Na 2 EDTA Buffer-saturated phenol Water-saturated chloroform Autoclaved, distilled water Absolute ethanol ( 20 C) 70% (v/v) ethanol DE-81 paper discs (Whatman DE-81 or equivalent) 1.5-ml microcentrifuge tubes Microcentrifuge (15,000 g) Water baths (37 C and 65 C) Scintillation fluid Scintillation counter Protocol for the Forward Reaction DNA or RNA to be labeled with the forward reaction must have a 5 hydroxyl terminus. If the nucleic acid contains a 5 phosphate group, it must be dephosphorylated with bacterial alkaline phosphatase (14) or calf intestinal alkaline phosphatase (17) prior to the labeling reaction. Use this protocol to P-label dephosphorylated nucleic acids. Because synthetic linkers usually do not have a 5 phosphate, they can be used directly in the forward reaction by following the protocol modifications discussed in the section entitled Phosphorylating Synthetic Linkers. The amount of DNA in the T4 polynucleotide kinase reaction is based on picomoles of 5 ends. For information on how to calculate picomoles of ends, see Additional Information. The forward reaction will yield specific activities of up to cpm/pmol ends. Avoid precipitating DNA from buffers containing ammonium salts prior to the kinase reaction, because high levels of ammonium ions inhibit T4 polynucleotide kinase. Protocol for Forward Reaction 1. Dissolve the DNA (5 pmol ends) in 16.5 µl of autoclaved, distilled water. 2. Add the following components to a microcentrifuge tube: Component Amount Final Concentration Dephosphorylated DNA fragments (5 pmol ends) 16.5 µl 200 pmol ends/ml Forward reaction buffer (5X) 5 µl 1X [γ- P]ATP (10 µci/µl) 2.5 µl 1 µci/µl 3. Add 1 µl of T4 polynucleotide kinase (10 units/µl). Mix by gentle pipetting. 4. Centrifuge the reaction briefly to collect the reaction mix at the bottom of the tube. 5. Incubate at 37 C for 10 min. 6. Spot 1 µl of the reaction mixture onto a DE-81 paper disc. Use this to determine the specific activity of the labeled DNA (see Analysis of End-Labeled DNA). 7. Stop the reaction by adding Na 2 EDTA to a final concentration of 5 mm or by heating the reaction to 65 C for 10 min. 8. Add 75 µl of autoclaved, distilled water. 9. Extract the reaction by adding 100 µl of buffer-saturated phenol and vortexing 10. Add 100 µl of water-saturated chloroform to the aqueous phase and vortex 11. Separate the labeled DNA from unincorporated nucleotides. (see Removal of Unincorporated Nucleotides).

3 3 Phosphorylating Linkers Linkers are double-stranded synthetic oligonucleotides used to introduce restriction endonuclease sites on the ends of blunt-ended DNA molecules. After digestion with the appropriate restriction endonuclease, the DNA is ligated to compatible termini in the vector. Most linkers have a 5 hydroxyl group, and require a 5 phosphate group to be added prior to ligation to the DNA. Several micrograms of linkers can be phosphorylated in one reaction. Since linkers are small, they have many more picomoles of 5 ends per microgram than large DNA fragments (1 µg of a 20-mer = 150 pmol 5 ends). The concentration of ATP in the reaction must be increased to ensure that each linker is phosphorylated. In general, linkers do not need to be radiolabeled, so there is no need to use [γ- P]ATP in the reaction. Phosphorylation of linkers can be monitored by performing a mock ligation and subsequent gel analysis to produce a ladder of linkers (5, 18) or by adding a radiolabeled tracer to determine counts per minute (cpm) incorporated (see Analysis of End-Labeled DNA). Protocol for the Exchange Reaction When DNA of high specific activity is not required, the exchange reaction is easier than the forward reaction because the DNA does not have to be dephosphorylated. The amount of DNA in the exchange reaction is based on picomoles of ends. Use this protocol to label the 5 ends of DNA with specific activities of to cpm/pmol of ends. Avoid precipitating DNA from buffers containing ammonium salts prior to the kinase reaction because high levels of ammonium ions inhibit T4 polynucleotide kinase. Phosphorylating Linkers 1. Reconstitute lyophilized linkers to a final concentration of 0.4 mg/ml in TE. 2. Add the following to a microcentrifuge tube: Component Amount Final Concentration DNA (2 µg) 5 µl 80 µg/ml Forward reaction buffer (5X) 5 µl 1X ATP (3 mm) 8 µl 1 mm autoclaved, distilled water 6 µl (up to 24 µl total volume) 3. Add 1 µl of T4 polynucleotide kinase (10 units/µl). Mix by gentle pipetting. 4. Centrifuge the reaction briefly to collect the reaction mix at the bottom of the tube. 5. Incubate at 37 C for 10 min. 6. Stop the reaction by adding Na 2 EDTA to a final concentration of 5 mm or by heating the reaction to 65 C for 10 min. 7. Add 75 µl of autoclaved, distilled water. 8. Extract the reaction by adding 100 µl of buffer-saturated phenol and vortexing 9. Add 100 µl of water-saturated chloroform to the aqueous phase and vortex 10. Add 0.1 volume of 3.0 M sodium acetate to the aqueous phase, followed by 2 volumes of absolute ethanol. 11. Centrifuge at 15,000 g for 30 min. Carefully remove supernate. 12. Dissolve the pellet in 10 mm Tris-HCl (ph 7.5), 0.1 mm Na 2 EDTA (TE). Protocol for Exchange Reaction 1. Dissolve the DNA to be labeled (5 pmol ends) in 14.5 µl of autoclaved, distilled water. 2. Add the following components to a microcentrifuge tube: Component Amount Final Concentration DNA (5 pmol ends) 14.5 µl 200 pmol ends/ml Exchange reaction buffer (5X) 5 µl 1X [γ- P]ATP (10 µci/µl) 5 µl 2 µci/µl 3. Add 0.5 µl of T4 polynucleotide kinase (10 units/µl). Mix by gentle pipetting. 4. Centrifuge the reaction briefly to collect the reaction mix at the bottom of the tube. 5. Incubate at 37 C for 10 min. 6. Spot 1 µl of the reaction mixture onto a DE-81 paper disc. Use this to determine the specific activity of the labeled DNA (see Analysis of End-Labeled DNA). 7. Stop the reaction by adding Na 2 EDTA to a final concentration of 5 mm or by heating the reaction to 65 C for 10 min. 8. Add 75 µl of autoclaved, distilled water. 9. Extract the reaction by adding 100 µl of buffer-saturated phenol and vortexing 10. Add 100 µl of water-saturated chloroform to the aqueous phase and vortex 11. Separate the labeled DNA from unincorporated nucleotides. (see Removal of Unincorporated Nucleotides).

4 4 Analysis of End-Labeled DNA The incorporation of radiolabel into the DNA can be monitored by trichloroacetic acid (TCA) precipitation of the labeled DNA or by adsorption of the labeled DNA to positively charged DE-81 paper that binds nucleic acids but not unincorporated nucleotides in 0.5 M Na 2 HPO 4. Although TCA precipitation is a commonly used method, it is restricted to DNAs or RNAs longer than 50 bases (19). Adsorption of the DNA to DE-81 filters, by comparison, has the advantage of binding oligonucleotides as well as longer DNAs. Use the following protocol to determine the amount of incorporated radiolabel from the T4 polynucleotide kinase labeling reactions: Analysis of End-Labeled DNA Use the DE-81 paper disc spotted with 1 µl of the T4 polynucleotide kinase reaction mixture (from step 6 of either labeling protocol). 1. Wash the disc six times by swirling it for 5 min in 10 ml of 0.5 M Na 2 HPO 4 per wash. 2. Wash the disc with distilled water for 2 min and then with absolute ethanol for 2 min. Air dry the disc at room temperature. This disc will be used to determine the P-label incorporated into the DNA. 3. Place the disc in scintillation fluid and count. Calculate the cpm incorporated per picomole of ends using the following equation: 1 total reaction volume cpm = cpm/pmol ends volume sampled pmol ends in reaction For example, 5 pmol ends were used in 25 µl total reaction volume. If cpm were obtained from 1 µl of the reaction, the cpm/pmol ends can now be calculated using the equation: 1 25 µl cpm = cpm/pmol ends 1 µl 5 pmol ends Removal of Unincorporated Nucleotides If necessary, the end-labeled DNA can be purified from reaction components by either ethanol precipitation or gel filtration chromatography. Complete removal of unincorporated nucleotides is generally unnecessary, so a simple ethanol precipitation (as described in chart to right) is usually sufficient. For purification of oligonucleotides 18 bases or shorter, or for more complete removal of unincorporated nucleotides, column chromatography or gel electrophoresis is necessary (20). Removal of Unincorporated Nucleotides 1. To the chloroform-extracted reaction, add 0.5 volume of 7.5 M ammonium acetate followed by 2.5 volumes absolute ethanol. Mix well by gentle inversion and place on ice for 30 min. 2. Centrifuge at 15,000 g at 4 C for 30 min. 3. Carefully remove the supernate. Warning: The supernate contains radiolabeled material and should be discarded properly. 4. Rinse the pellet with 70% (v/v) ethanol, and spin briefly. 5. Remove the supernate. Store at 20 C in 70% ethanol until ready to use. 6. When ready to use, centrifuge at 15,000 g at 4 C for 5 min. Discard supernatant. Briefly air dry pellet and resuspend in an appropriate buffer.

5 5 Troubleshooting Some possible causes of inefficient phosphorylation when using T4 polynucleotide kinase are described below, accompanied by suggested solutions. Possible Cause Incorrect radioactive isotope was used. Suggested Solution Use [γ- P]ATP. Only the γ-phosphate can be transferred to the DNA. Use ATP supplied as an aqueous solution. If ethanol is present, lyophilize the ATP solution in the reaction tube, and add autoclaved, distilled water to the volume recommended in the protocol. Use ATP with a specific activity of at least 3,000 Ci/mmol. Use ATP within a few days upon arrival from the supplier. Isotope of very high specific activity breaks down quickly, yielding degradation products that may interfere with the labeling reaction (21). Inhibitors were present. Avoid inhibitors such as SDS, ammonium ions, or phosphate ions (see Additional Information). Ethanol precipitate or purify the DNA by column chromatography to remove inhibitors. Avoid ethanol precipitation of the DNA with ammonium ions prior to the labeling reaction. Use sodium acetate as the precipitation salt. Avoid performing the reaction in the presence of agarose including low melting point agarose. Even low levels of agarose inhibit T4 polynucleotide kinase (22, 23). Contaminating low-molecular-weight nucleic acids were present Avoid using trna as a carrier prior to the labeling reaction. Small oligonucleotides or trna can contribute a large number of 5 ends even though they make up only a small percentage of the total weight. Incorrect reaction conditions were used. Use the buffer and nucleotide concentrations specified. If the DNA has a 5 hydroxyl group, use the forward reaction. If the DNA has a 5 phosphate, use the exchange reaction or dephosphorylate the DNA and use the forward reaction. Use the number of picomole ends that are recommended in the protocols. When using a greater number of picomole ends than recommended, scale up the reaction proportionally.

6 6 Additional Information Determination of picomoles of 5 ends Use the following formulas to determine the picomoles of 5 ends of the DNA to be used in the T4 polynucleotide kinase reaction: For double-stranded DNA µg DNA 10 6 pmol pmol 5 ends/assay = 2 MW µmol MW = (number of base pairs) (660 µg/µmol) (assume 50% A + G content) For single-stranded oligonucleotides µg DNA pmol 5 ends/assay = 10 6 pmol MW µmol MW = (number of base pairs) (330 µg/µmol) (assume 50% A + G content) Radiolabeling Oligonucleotides Oligonucleotides can be labeled at the 5 end using T4 polynucleotide kinase. To yield oligonucleotides with high specific activity (~ cpm/pmol ends), the amount of [γ- P]ATP in the reaction must be increased to compensate for the increased picomoles of ends per microgram of DNA (24) (1 µg of a 20-mer = 150 pmol 5 ends). Collins and Hunsaker (24) recommend a reaction containing 200 µci of isotope, 100 ng of a 17- to 19-mer oligonucleotide, 2 µl of 5X forward reaction buffer, and sufficient autoclaved, distilled water for a final volume of 9 µl. The isotope is lyophilized to keep the volume small. One microliter of T4 polynucleotide kinase is added to initiate the reaction, which is incubated for 30 min at 37 C. Alternatively, hybridization sensitivity of oligonucleotide probes can be improved by labeling the 3 end with terminal deoxynucleotidyl transferase (24, 25). Labeling RNA T4 polynucleotide kinase will end-label RNA (26, 27) by the forward reaction once it has been dephosphorylated. The exchange reaction will not recognize 5 -triphosphates as substrates. In 5 end labeling reactions, GIBCO BRL T4 Polynucleotide Kinase caused no detectable breakdown of 5S RNA (28). Labeling Blunt-Ended DNA or DNA with 3 Extensions Labeling recessed ends or blunt ends can be improved by heating the DNA at 100 C for 2 min and quick chilling on ice to denature the DNA. Then, perform the reaction immediately. Addition of polyethylene glycol (PEG) can also improve the efficiency of labeling (see Addition of Polyethylene Glycol). Alternatively, for some applications, the 3 end can be labeled with terminal deoxynucleotidyl transferase as opposed to labeling the 5 end with T4 polynucleotide kinase (24, 25). Inhibitors Ammonium ions at 7 mm will inhibit T4 polynucleotide kinase by 75% (1). T4 polynucleotide kinase in a 70-mM sodium or phosphate buffer will have 5% of the activity as in a Tris buffer (1). Agarose at 0.002% inhibits T4 polynucleotide kinase activity by 17% (22, 23). Dextran sulfate at 0.06 µg/ml will cause 80% inhibition while 20 µg/ml of dextran shows no inhibition of the enzyme. Polysaccharide sulfates were found to inhibit T4 polynucleotide kinase whereas the nonsulfated polysaccharides did not (22, 29). Phosphate ions (2, 29), agar (22), and trace amounts (0.001%) of SDS (26) also inhibit T4 polynucleotide kinase. Isotopes [γ- 35 S]ATP is a very poor substrate for T4 polynucleotide kinase and is not recommended for 5 end labeling. Addition of Polyethylene Glycol The addition of the high molecular weight polymer PEG 8000 improves the efficiency and rate of the T4 polynucleotide kinase reaction for DNA and RNA without changing the nature of the products. For labeling recessed 5 termini or for very low concentration of substrate, the addition of 4% to 6% (w/v) PEG 8000 improves phosphorylation (21, 30). However, PEG does not have an effect on the phosphorylation of single-stranded oligonucleotides in either the forward or exchange reactions (21). Specific Activity of the Probe The specific activity of end-labeled DNA can be improved by increasing the amount of [γ- P]ATP or decreasing the picomoles of ends in the reaction (31).

7 7 References: 1. Richardson, C.C. (1972) in Progress in Nucleic Acids Research (Cantoni, G. L. and Davies, D. R., eds.) Vol. 2, p. 815, Harper and Row, New York. 2. Richardson, C.C. (1965) Proc. Natl. Acad. Sci. USA 54, Cameron, V. and Uhlenbeck, O.C. (1977) Biochemistry 16, FOCUS (1983) 5:3, Life Technologies, Inc. (1994) Focus on Applications: T4 DNA Ligase, Technical Bulletin Chaconas, G. and van de Sande, J.H. (1980) Methods Enzymol. 65, Maxam, A. and Gilbert, W. (1980) Methods Enzymol. 65, Conner, B.J., Reyes, A.A., Morin, C., Itakura, K., Teplitz, R.L., and Wallace, R.B. (1983) Proc. Natl. Acad. Sci. USA 80, Zeff, R.A. and Geliebter, J. (1987) FOCUS 9:2, Albretsen, C., Haukanes, B., Aasland, R., and Kleppe, K. (1988) Anal. Biochem. 170, Struhl, K. (1989) in Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., eds.), Vol. 1, p , Greene Publishing Associates and Wiley-Interscience, New York. 12. Berk, A.J. and Sharp, P.A. (1977) Cell 12, Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), p , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 14. Life Technologies, Inc. (1989) Focus on Applications: Bacterial Alkaline Phosphatase, Technical Bulletin Berkner, K.L. and Folk, W.R. (1977) J. Biol. Chem. 252, Midgley, C.A. and Murray, N.E. (1985) EMBO J. 4, Tabor, S. (1989) in Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., eds.), Vol. 1, p , Greene Publishing Associates and Wiley-Interscience, New York. 18. Wu, R., Wu, T., and Ray, A. (1987) Methods Enzymol. 152, Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), p. E.18, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 20. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), p , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 21. Harrison, B. and Zimmerman, S.B. (1986) Anal. Biochem. 158, Wu, R. (1971) Biochem. Biophys. Res. Commun. 43, Tabor, S. and Struhl, K. (1989) in Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K., eds.), Vol. 1, p , Greene Publishing Associates and Wiley-Interscience, New York. 24. Collins, M.L. and Hunsaker, W.R. (1985) Anal. Biochem. 151, Life Technologies, Inc. (1990) Focus on Applications: Terminal Deoxynucleotidyl Transferase, Technical Bulletin Cannistraro, V.J., Strominger, M.B., Wice, B.M., and Kennell, D.E. (1985) J. Biochem. Biophys. Methods 11, Cannistraro, V.J., Wice, B.M., and Kennell, D.E. (1985) J. Biochem. Biophys. Methods 11, Cannistraro, V.J. and Kennell, D. (1987) FOCUS 9:2, Lillehaug, J.R. and Kleppe, K. (1975) Biochemistry 14, Harrison, B. and Zimmerman, S.B. (1986) Nucl. Acids Res. 14, Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), p , Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

8 8 Ordering Information Product Cat. No. Size T4 Polynucleotide Kinase units ,000 units T4 Polynucleotide Kinase Forward Reaction Buffer ml T4 Polynucleotide Kinase Exchange Reaction Buffer ml Related Products Cat. No. Size 5 DNA Terminus Labeling System reactions Bacterial Alkaline Phosphatase (BAP) ,500 units Calf IntestinalAlkaline Phosphatase (CIAP) ,000 units Calf Intestinal Alkaline Phosphatase (CIAP) Buffer Set ml each Terminal Deoxynucleotidyl Transferase units units Phenol g g g kg Buffer-Saturated Phenol ml ml ml ml Tris g kg kg g Tris Hydrochloride g For research use only. Not for diagnostic or therapeutic use in humans or animals. TECH-LINE SM and the Life Technologies logo are marks of Life Technologies, Inc. 95-0