Figure 4.

Processivity assay using a heteropolymeric HIV PBS RNA template. Processivity of the recombinant HIV-1 subtype B and C RT enzymes was assessed using heteropolymeric HIV PBS RNA template and D25 DNA primer. The DNA primer D25 was labeled with ³²P at the 5'-terminus and annealed to the RNA template at an equimolar ratio. Processivities were analyzed by monitoring the size distribution of DNA products in fixed-time experiments at three different concentrations of dNTPs in the presence of heparin trap (+ Trap). Parallel reactions were run in the absence of trap (- Trap) at 200 μM of dNTPs to ensure that similar amounts of enzyme activities were present in the reactions. The sizes of some fragments of the ³²P-labeled 25bp DNA ladder (Invitrogen) in nucleotide (nt) bases are indicated on the left side of the panel. All reaction products were resolved by denaturing 6% polyacrylamide gel electrophoresis and visualized by phosphorimaging. Trap control: control reactions in which the heparin trap was preincubated with substrates before the addition of RT enzymes. T/P: control reaction in which no RT enzymes were included. Positions of ³²P -labeled D25 primer (³²P -D25) and the 471-nt full-length extension DNA (FL DNA) product are indicated on the right.