Figure 3.

3′-end labeling of DNA and RNA oligomers using Terminal deoxynucleotidyl transferase (Tdt). (A) DNA or RNA oligomers are incubated with Tdt in the presence of labeled deoxynucleotides, which are added to the 3′-end of the DNA or RNA oligomers by the Tdt. Usually, more than one labeled deoxynucleotide is added to the DNA oligomers. Previously, the addition of multiple labeled deoxynucleotide was reported for the RNA oligomers (75). (B) An agarose gel of Tdt labeling reactions in the presence of DNA or RNA oligomers and varying concentrations of dTTP, dUTP or digoxigenin-labeled dUTP (dig-dUTP). DNA or RNA oligomers were incubated with Tdt and respective deoxynucleotide in the 1× Tdt reaction buffer for 1 h at 37°C (Materials and Methods section). The reactions were stopped with the addition of EDTA and denatured with urea prior to loading them on a denaturing 20% PAGE. Lanes: M, single-stranded DNA marker. The 20 and 30 nt long oligomers are indicated; 1, DNA oligomer incubated in the absence of deoxynucleotides; 2, DNA oligomer labeled with 50 μM dig-dUTP; 3, DNA oligomer extended at its 3′-end with 50 μM dTTP; 4, RNA oligomer incubated in the absence of deoxynucleotides; 5, RNA oligomer labeled with 50 μM dig-dUTP; 6, RNA oligomer labeled with 250 μM dig-dUTP; 7, RNA oligomer incubated with 50 μM of dTTP; 8, RNA oligomer incubated with 10 mM dTTP.