Platinum superfi high fidelity dna polymerase

Unless stated otherwise, all the described reactions in this paper were carried with the described products according to the manufacturer’s instructions: gel purifications were performed using Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI, USA); beads purifications were performed using AMPure XP beads (Beckman Coulter, Brea, CA, USA); concentrations were determined using Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA); reverse transcription (RT) reactions were performed using SuperScript III or IV Reverse Transcriptase (Thermo Fisher Scientific); polymerase chain reactions (PCR) were made using Platinum SuperFi high-fidelity DNA Polymerase (Thermo Fisher Scientific) or Q5 high-fidelity DNA Polymerase (New England Biolabs (NEB), Ipswich, MA, USA).

PCRs were performed with a SimpliAmp thermal cycler (Thermo Fisher Scientific) using Platinum SuperFi high-fidelity DNA polymerase (Thermo Fisher Scientific). The resulting PCR fragments were purified with a NucleoSpin gel and PCR cleanup kit (Macherey-Nagel) according to the manufacturer’s instructions.
PCR products purified with the NucleoSEQ kit (Macherey-Nagel) were sequenced on both strands using the BigDye Terminator (version 3.1) cycle sequencing kit from Applied Biosystems and the ABI Prism 310 genetic analyzer.

The HCV E1 and E2 genes were amplified by nested PCR using Platinum SuperFi High Fidelity DNA Polymerase (Thermo-Fisher scientific). The first round of PCR was performed using 4 μL of RT reaction product and a set of external primers (5'-ACATGAAGCTTCGCCGACCTCATGGGGTACA -3', 5'-CTCCGGATATCGCAGCCATCTCCCGG TCCAT-3'). After an initial denaturation step of 98°c for 3 min, 13 cycles of 98°c for 30sec, 60°c for 30 sec and 72°c for 2 min. After cycling, a final extension period at 72°c for 5min was added. The second round of PCR was performed using 4μL of the first PCR reaction and a set of internal primers (5'-GTGAACTATGCAACAGGGAA-3', 5'-CAGAAGA ACACAAGGAAGGAGAG-3'). We used the same PCR protocol for both rounds, while running 27 cycles for the second round. After verifying the amplification success using gel electrophoresis, the nested PCR product was purified using AMPure XP system (Beckman Coulter Life Sciences) at 0.5X ratio. 60 μg of DNA was sequenced using Sanger sequencing (Hylabs). During sequencing, at each position the nucleotide presenting maximal signal peak is assigned. In some positions, signals with equivalent strengths were received for two bases (“double peaks”), thus prompting a random choice between them. This could have detrimental effects on the accuracy of phylogenetic inference, therefore such positions were omitted from analysis.