Measuring fitness and recombination frequency in the multiple cycle system. U87.CD4.CXCR4 cell cultures dually infected with two isolates of different subtypes (A+D; panel A) or the same subtype infections (A+A or D+D; panel B) and then harvested for analyses. Subtype or isolate-specific primers were employed to amplify parental or recombinant HIV-1 env DNA (X axes) from specific dual infections (Z axes). Copy numbers on the Y axes were derived from control PCR amplifications with known copy numbers of subtype A and D DNA templates (102 to 108 copies/reaction) (see Materials and Methods). Relative fitness values (W) and frequencies of recombination from these dual infections/competitions were calculated as described in the inset of panel C. Briefly, conserved primers were utilized to PCR amplify the env genes from parental and recombinant env progeny from each dual infection to measure fitness by HTA[54,62,63]. These PCR products were then denatured and annealed to a radiolabeled env probe, which was amplified from a subtype E HIV-1 env clone (E-pTH22. DNA heteroduplexes specific for the each parental isolate were resolved on a 6% nondenaturating polyacrylamide gel. A sample autoradiograph and calculations of relative fitness is defined in panel D. A plot of the fitness differences (WD = Wmore fit/Wless fit) or of percent recombinants (right Y axis) for each dual infection pair is shown in panel E.