Fig. 2.

Reconstruction of residual plasma HIV-1 genomes as DNA form. Panel a schematically shows the corresponding strategy. The small 337 bp (R-gag) and 408 bp (U3-R) DNA fragments and the large 3.5 kb, and 5.3 kb fragments were separately amplified by RT-nested PCR using residual plasma vRNAs as targets. Each horizontal small arrow indicated by A through H represents a pair of primers (Additional file 5: Table S1) used in the RT-nested PCR. The U3, R and U5 regions are the elements of the HIV-1 long-terminal repeat (LTR). R-gag and U3-R fragments were fused to generate the U3-gag fragment (not shown), which was later combined with 5.3 kb and 3.5 kb fragments to build the 5′- and 3′-halves, respectively (see Additional file 1). Panels b, c and d show the amplified fragments (indicated by arrows) in agarose gels. Lanes are marked with the corresponding fragment names. Lane M shows the GeneRuler 1 kb plus DNA ladder (Thermo Scientific). In panel d, the size difference between the R-gag and U3-R bands is not reflected in the agarose gel due to anomaly in the migration of DNA bands, but the identity of these bands was verified by sequence analyses. A 200 bp band seen below the 337 bp R-gag band is not HIV-specific