γ 32p atp 5 triphosphate

T4 polynucleotide kinase (PNK) and γ-³²P-ATP-5′-triphosphate were obtained from Perkin Elmer. Oligonucleotides were labeled by mixing PNK, PNK buffer, ATP, DNA, and water (final volume = 50 μL) according to manufacturer’s procedure followed by incubation at 37° C for 45 min. Radiolabeled materials were passed through a G-25 sephadex column followed by purification via electrophoresis (20% denaturing PAGE). The bands of interest (slowest migrating) were extruded, crushed, and soaked in a saline buffer solution (0.1 M NaCl) for 12 h at 37° C. The remaining solution was filtered and concentrated to dryness under reduced pressure followed by precipitation in a NaOAc (300 mM) / ethanol 1:5 solution (by volume) using a dry ice/ethanol bath. Supernatant was removed and the remaining oligonucleotide was concentrated under reduced pressure and dissolved in water. Activity was assessed using a Beckmann LS 6500 scintillation counter. Electrophoresis was not necessary for DNA strands previously purified via HPLC (purchased from manufacturer). A tracking dye (90% formamide containing 0.05% xylene cyanol and 0.05% bromophenol blue) was added to a well on each side of the gel; and this procedure was repeated for all subsequent experiments.