Figure 3.

NC-mediated annealing and RT extension using the HIV-1 MAL template. (A) Polyacrylamide gel showing the results of NC-mediated annealing of ³²P-labeled tRNA primers to the 311-nt template. The − and + signs represent the absence and presence of NC in the reactions. The identity of each primer/template combination used is indicated above the lanes. (B) Agarose gel (2%) showing PCR-amplified primer extension products. The tRNA primers were extended by either 40 nM (lane 3) or 400 nM (lanes 2 and 4) RT. Lane 1, DNA size marker; the length indicated is in base pairs. Lanes 2–4, extension was carried out in the absence (−) or presence (+) of tRNA^Lys-3 primer using a template complementary to either human tRNA^Lys-3 (Lys) or E. coli tRNA^Pro-3 (Pro). (C) DNA sequence of the PCR product obtained in lane 4 (B). Nucleotides corresponding to the E. coli Pro-PBS are explicitly shown. The choice of sequencing primer resulted in obtaining the sense sequence of the RNA template and the antisense sequence of tRNA^Lys-3. The position of nucleotides G1 and U67 of tRNA^Lys-3 are shown in italics to indicate that they are complementary to the sequence shown in the gel.