Figure 2
PCR-based lacZα-complementation system used to determine the fidelity of HIV RT. (A) An overview of the procedure used to assess polymerase fidelity is presented. RNA is represented by broken lines and DNA is represented by solid line. Primers have arrowheads at the 3′ end. The ~760 nt template RNA used as the initial template for HIV RT RNA-directed DNA synthesis is shown at the top with the 3′ and 5′ ends indicated. The positions of PvuII and EcoRI restriction sites are indicated for reference to the vector. The filled box at the bottom of the figure is the 115 base region of the lacZα gene that was scored in the assay. Details for specific steps are provided under Materials and Methods. (B) Plasmid pBSM13ΔPvuII1146, is shown. Relevant sites on the plasmid are indicated and numbering is based on the parent plasmid (pBSM13+ (Stratagene)). (C) The nt and amino acid sequence for the 115 base region of the lacZα gene that was scored in the assay is shown. Both strands of the DNA plasmid are shown since HIV RT synthesis was performed in both directions (see Figure 2A). A line is drawn above the 92 nts that are in the detectable area for substitution mutations while frameshifts can be detected over the entire 115 nt region. Based on a previous cataloging of mutations in this gene [51], the assay can detect 116 different substitutions (33.6% of the 345 possible substitutions in the 115 nt sequence) and 100% of the frameshift mutations.