Sequencing of HIV-1 polymerase (pol) (protease and reverse transcriptase genes)
HIV RNA was extracted from plasma using the QIAmp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) and reverse-transcribed into complementary DNA (cDNA) using the RETROscript kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. A nested polymerase chain reaction (PCR) was performed using 10 μL of diluted cDNA or cellular DNA template added to 40 μL of the reaction mixture for the first round. The reaction mixture comprised 5.0 μL of 10× PCR buffer containing magnesium chloride and 1.0 μL of 10 nM deoxynucleotide triphosphate Mix (GeneAmp, Applied Biosystems, Foster City, CA, USA), 0.25 μL of Taq DNA Polymerase (Roche Diagnostics, Indianapolis, IN, USA), 31.75 μL of molecular grade water, and 1 μL of each of the 20 μM primers. The 50-μL samples were heated to 94 °C for 2 min and subjected to 35 cycles of 30 s at 94 °C, followed by 30 s at 52 °C and 60 s at 68 °C. Subsequently, the samples were heated to 68 °C for 10 min and stored at 4 °C until use. The second round of PCR was performed using 5 μL of the product from the first round as the template, which was added to 45 μL of the reaction mixture to obtain a final volume of 50 μL. This reaction mixture comprised the same reagents, with the volume of molecular-grade water increased to 36.75 μL. The thermal cycling parameters were: 35 cycles of 30 s at 94 °C, followed by 30 s at 55 °C and 60 s at 72 °C. The PCR products from each sample were sequenced using Prism Dye terminator kits (ABI) on an ABI 3730 DNA Analyzer (Applied Biosystems, Foster city, CA, USA).