PCR optimizations

Ribonucleic acid and DNA PCR reactions for the HIV-1 pol region were set-up with in-house protocols as shown in appendix III at 50°C annealing temperatures for both RNA and DNA reactions. PCR conditions for the HIV-1 pol region were optimized for cDNA synthesis and first round PCR initially with SuperScript™ III One-Step RT-PCR System with Platinum® taq polymerase High Fidelity enzyme (Life Technologies, Invitrogen) and components were pipetted as indicated in appendix III. Template RNA concentrations were increased by 5 µl for each trial as shown in appendix III. Primer sequences were as indicated in the table 4. A nested PCR reaction with the verbatim high fidelity DNA polymerase enzyme (Thermo Scientific, Darmstadt, Germany) was set-up as indicated in appendix III. The PCR primer sequences for the second round reaction are given in table 4 above. The PCR consisted of 40 cycles with 1°C increments in annealing temperature of up to 60°C. Upon gel electrophoresis, no PCR band was realized although viral load of samples used was ≥ 500,000 copies/ml of plasma. Nested PCR for DNA samples were carried out with the same primers pairs and first and second round PCRs were done as shown in appendix III, but there were also no PCR bands found on agarose gels. Note that only one condition was changed at a time for each round of optimization.

Upon recommendations from a more experienced lab member who also worked on a similar project, primers were changed to the following combinations: cDNA and first round synthesis; 2.5 µl of 10 µM/µl each of Pol1 forward and RT-rev-stu1 reverse primers and the second round nested PCR reaction contained the same amounts of primer concentrations for Prot-for-EcoR1 forward primer and RT4 reverse primer as given in table 4. Nested PCR conditions were the same as shown in appendix III. No bands were seen on agarose gel photographs. PCR reagents were changed to the phusion high fidelity DNA polymerase (New England BioLabs, Frankfurt, Germany) and the same PCR conditions were repeated with the same primers and primer concentrations as mentioned above with 10 µl of 5x Phusion high fidelity or GC rich buffer but again no bands were seen after taking gel photographs.

Materials and Methods

42 Polymerase chain reactions were again tried with various primer combinations as shown in appendix III using the phusion high fidelity DNA polymerase (New England BioLabs, Frankfurt, Germany), the same PCR cycling conditions as described in the paragraph above. This procedure also yielded no results.

Three plasma samples were selected and tried on the ViroSeq HIV-1 Genotyping Kit (Abbott GmbH & Company, Wiesbaden, Germany) at the Diagnostic section of the University hospital, Heidelberg Germany following the manufacturer’s protocol and amplification was sucessful. The experiment therefore proved that patient RNA samples were still viable for PCR reactions. In order to preserve patient samples for the actual experiments, 10 plasma samples from HIV infected patients from the diagnostic unit of the Heidelberg University Hospital were obtained and RNA extractions were done with the ViroSeq HIV-1 RNA extraction Kit (Abbott GmbH & Company, Wiesbaden, Germany) and the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). PCR was again repeated with the ViroSeq HIV-1 Genotyping Kit which had its own primer sets (Abbott GmbH & Company, Wiesbaden, Germany) and the SuperScript™ III One-Step RT-PCR System with Platinum® Taq polymerase High Fidelity enzyme (Life Technologies, Invitrogen) and the Phusion High Fidelity DNA Polymerase PCR kit (New England BioLabs, Frankfurt, Germany). The ViroSeq protocol worked but the SuperScript RT- PCR System with the Phusion PCR protocols did not work. The annealing temperature was at 55°C for 45 minutes during the cDNA synthesis and at 55°C for 30s for both first and second round DNA synthesis. The ViroSeq primers could not be used with my PCR protocol because the primer sequences and concentrations were unknown.

As a proof of concept, further proceeded by using previously designed primers sets that specifically amplify a 400bp region spanning the 5’ end of HIV-1 RT to amplify the Burkina Faso samples (Tebit et al., 2006). These primers were tried again on 5 pairs of RNA samples extracted with the ViroSeq HIV-1 RNA extraction Kit (Abbott GmbH & Company, Wiesbaden, Germany) and the QIAamp Viral RNA Mini Kit. PCRs were performed with the combination of the SuperScript RT-PCR System with the Phusion PCR protocols using the same conditions as described in the previous paragraph and all 5 pairs of samples were amplified. This suggested that the PCR primers used previously were not working and had to be changed. The primers were BFp1 for and BFp4b rev were first round primers and BFp3 for and BFp4c rev were second

Materials and Methods

43 round primers (table 4). Although these set of primers worked well, they could not be used to amplify the expected region for the purpose of this study.

New sets of HIV-1 pol primers were designed with the help of Stefan Seitz from the Molecular Virology Department of the UniversitätKlinikum, at the Otto Meyerhof Centre in the Heidelberg University Hospital. We designed HIV-1 sequences encoding approximately 1300bp of gag (p6), PR and about 255 aa in RT. These primers were designed based on consensus sequences of an HIV-1 CRF02_AG subtype isolated in 2006 with the GenBank accession number AB231895. These primer pairs were namely New F1 and New R1 used in the first round PCR reaction and New F2 and New R2 used in the second round PCR reaction. These primers were used together with the SuperScript RT-PCR System and Phusion PCR protocols, which gave non-specific bands including the band of interest on agarose gel. Gradual 1°C increments of annealing temperatures and 30s increments on annealing times did not resolve the problem. Activation of PCR reactions at 95°C for 5 min to denature pre-annealed primers, before addition of the polymerase also did not resolve the problem.

Since the protocol described above did not work efficiently, new PCR protocols were adapted from Kousiappa et al 2009. The protocol did work along with its published primers (table 4) and with minor modifications as described in section 2.11. The amplified region was about 1460bp of the HIV-1 pol region.