Quantifying HIV Proviral DNA Levels

To determine copy numbers of HIV proviral DNA, a nested PCR method was used. The first PCR was performed using a forward primer (Alu-F, 0.2 μM) that targeted genomic alu sequences randomly located near integrated proviruses and an HIV-specific gag reverse primer (Gag-R, 1.2 μM). Other PCR conditions were 200 μM of each dNTP, 1X LongAmp taq buffer (NEB), 5 units of LongAmp taq DNA polymerase (NEB) and 50 ng of DNA sample extracted from infected cells in a 50 μL final reaction volume. Amplification was performed using the following thermocycler program: initial activation heating 94 °C for 2 min, followed by 20 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and extension at 65 °C for 3 min and a final extension reaction at 65 °C for 10 min. The PCR product is diluted 20-fold and 5 μL of the diluted PCR product is used as an input material for the second PCR reaction, which is performed using LRT forward and reverse primers and probe using the Rotor-Gene Probe PCR kit (Qiagen) as described above. Proviral copy numbers were determined using the J-lat cell integration standard as previously described [21 (link), 31 (link), 32 (link)]. Genomic DNA copy numbers were determined using the RPP30 reference gene using the same amount of input DNA used to measure proviral copy numbers.