cDNA synthesis, PCR amplification and purification

Reverse transcription was performed by using Superscript III First-strand synthesis System for RT-PCR (Invitrogen 18080–051). The cDNA was amplified by touchdown (TD) polymerase chain reaction (PCR) procedure with 2 rounds of amplification. Each amplification run was performed in a 50μl reaction volume using Promega PCR master mix (Promega, Madison, WI, USA cat M7505) supplemented with 0.4μM of primer Table 1. Cycling conditions for both outer and nested TD-PCR was as follows: initial incubation at 95°C for 3 min followed by 10 cycles at 95°C for 30 s, 64°C for 30 s with a 1°C decrement per cycle, and 72°C for 50 s. Subsequently, 25 cycles were performed at 95°C for 30 s, 55°C for 30 s, and 72°C for 50 s followed by a final extension of 5 min at 72°C. The second round PCR amplicons were separated by gel electrophoresis. The expected PCR product size of about 850 pb were excised from the gel and purified using QIAquick Gel Extraction Kit (Qiagen).

(+), forward primer; (-) Reverse primer;

^a Position according to the HXB2 coordinates