Preparation of end-labeled oligonucleotides

The 3’-biotin labeling of oligonucleotides was performed with terminal deoxynucleotidyl transferase (Invitrogen; Carlsbad, CA) and biotin-16-ddUTP (Roche; Indianapolis, IN) using the following conditions. Three hundred nanograms of each sense strand oligonucleotide was incubated at 37°C for 30 min in 50 μl reaction mixture containing 15 units of deoxynucleotidyl transferase, 2 μM biotin-16-ddUTP, 0.1 M potassium cacodylate (pH 7.2), 2 mM CoCl₂, and 0.2 mM dithiothreitol. After this incubation, 1 μl of 0.5 M EDTA (pH 8.0) and 50 μl of chloroform:isoamyl alcohol (24:1) were added to the reaction mixture. The mixture was centrifuged at 13,000xg for 3 min, and then the upper phase was recovered and precipitated with ethanol. The 3’-biotin labeled sense-strand oligonucleotides were annealed to unlabeled anti-sense-strand oligonucleotides. For the 5’-³²P labeling, one microgram of each oligonucleotide was phosphorylated at 37°C for 1 hour with T4 polynucleotide kinase (New England Biolabs; Ipswich, MA) in the presence of [γ-³²P]ATP and purified on G-50 spin columns (Roche; Indianapolis, IN). The 5’-³²P labeled sense-strand oligonucleotide was annealed to an unlabeled oligonucleotide representing the opposite strand.