RT PCR services and products of the right size were regularly obtained for the substantial fragment and two overlapping fragments in plasma samples with 1,000 copies/ml of HIV RNA. Not surprisingly, a greater success rate in PCR amplification was observed with both overlapping fragments than with the large 3. 4 kbp solution, especially when plasma samples with viral loads between 50 and 1,000 ubiquitin conjugating copies/ml were used. Extremely reproducible achievement in RT PCR amplification of the services and products was received when 20 plasma samples were examined with different viral loads. Information on this test using two different operators with different lots of essential reagents and over a 7 day period are described in Table S1 of the supplemental material. Finally, the specificity of the different RTPCR primers and reaction mixtures was examined using nucleic acids from a series of RNA and DNA viruses. As expected, no cross reactivity was observed with any of those viruses as all RT PCRs, either for the large fragment or the 2 organic chemistry smaller overlapping pieces, failed to create any detectable amplicons. Development of 3 Gag/PR/RT/INT recombinant viruses. Unlike previous approaches that use homologous recombination in mammalian cells or ligation based cloning methods to produce recombinant viruses, here we used a yeast based recombination/ gap repair method to present individual derived HIV sequences in to a vector, together with the final goal of making reproduction qualified chimeric viruses. As explained above, a large HIV genomic region from the Gag protein p2 to the integrase coding region was RT PCR amplified as a large fragment or two overlapping fragments from plasma samples or HIV 1 isolates. The p2 INT recombinant viruses were then made by recombining the huge fragment or two overlapping PCR services and products in to a near full-length HIV 1 genome vector. The URA3 gene was taken for the p2 INT HIV 1 coding sequence in the not quite full-length NL4 3 vector, i. e., pRECnfl TRP p2 INT/URA3. This vector was made to express the Renilla order CX-4945 luciferase gene between the nef coding regions and env without affecting the expression of any HIV gene, as previously described. As described above plasmid DNA was isolated from yeast colonies and utilized to transfect HEK293T cells after a number of intermediate microbial measures. Recombinant viruses were collected at 2 times posttransfection and characterized viral stocks used in drug susceptibility assays based on the disease of MT 4 cells in the existence of serial dilutions of antiretroviral drugs and quantifying virus replication by measuring the activity of Renilla luciferase. Eventually, world wide sequencing confirmed that HIV 1 sequences of the first plasma samples and the corresponding p2 INT recombinant viruses were nearly identical. Effectiveness of the novel drug vulnerability assay.