Amplification of HIV Pol

The HIV Pol (PR and RT) region of 1.1 kb, spanning the entire protease and the first 800 bps of RT, was amplified with a transcriptor one-step RT-PCR kit (Roche Applied Science, Penzberg, Germany). A one-step RT-PCR reaction mixture was prepared to consist of 0.5µL Transcriptase Enzyme Roche One Step, 7µL of RNase-free water, 5µL of transcriptor one-step RT-PCR 5X Buffer, 2.5µL mixture of primers CWF1-LNA2(5’-GAA GGA CCA AAT GAA AGA YTG-3’ (2 μM) and CWR1-LNA3 (5’-GCA TAC TTY CCG TTT TCA G-3’ (2 μM), and 10µL of HIV RNA, making a total of 25µL. The PCR parameters for the initial one-step PCR were reverse transcribed at 50°C for 30 minutes, initial denaturation at 94°C for 7 mins, 10 cycles consisting of a denaturation stage at 94°C for 10 seconds, annealing stage of 55°C for 30 seconds and an extension step of 68°C for 2 minutes and then 35 cycles with denaturation at 94°C for 10 seconds, annealing stage of 55.5°C for 30 seconds and final extension step of 68°C for 2 minutes, increasing each cycle by 10 seconds. The last stage was the final elongation at 68°C for 5 minutes with a hold stage at 4°C for a maximum of 18 hours. The nested PCR was performed using 1µL aliquot of first-round PCR product mixed with 9.0µL of RNase-free water, 12.5µL of Phusion High-Fidelity PCR Master Mix with HF Buffer, and 2.5µL of primers CWF1-LNA2(5’-GAA GGA CCA AAT GAA AGA YTG-3’ (2 μM) and RT-20C (5’CTG CCA ATT TCT AAC TGC CTT C-3 ‘(2 μM), making 25µL of reaction volume. The PCR parameters for the nested PCR were initial denaturation at 98°C for 30 seconds, 35 cycles made of denaturation at 98°C for 10 seconds, an annealing stage of 62°C for 30 seconds, an extension step of 72°C for 20 seconds, and the final elongation at 72°C for 10 minutes with a hold stage at 4°C for a maximum of 18 hours. Amplification was confirmed by electrophoresis in a 1% agarose gel in Tris-Borate-EDTA (TBE) buffer and ran at 90 volts for 45 minutes stained with 5μL ethidium bromide (0.5 mg/mL) and visualized under a UV source (260 nm). Failed samples were re-extracted and PCR amplification was repeated once using a set of rescue primers (Table 1) with the same PCR parameters and conditions.

Rescue Primers Used When Samples Failed to Amplify

Note: **A mixture of primers F1a and F1b.