Tempase hot start dna polymerase

Plasmodium falciparum merozoite surface protein 2 (Pfmsp2) genotyping was performed on extracted gDNA from parasites at day 0 and at day 4, as previously described [15 (link)], with the following modifications: the conditions of the outer PCR were as follows: 94 °C for 15 min, 30 cycles of 94 °C for 1 min, 58 °C for 2 min and 72 °C for 2 min, followed by 58 °C for 2 min and 72 °C for 5 min; for the nested PCR reactions: 94 °C for 15 min, 30 cycles of 94 °C for 30 s, 61 °C for 1 min and 72 °C for 1 min, followed by 58 °C for 2 min and 72 °C for 5 min. The PCR reactions contained 0.00625 µM of the outer primer pairs included in the outer PCR and 0.125 μM of the primer pairs for the nested PCRs, 1:1 of TEMPase Hot Start DNA Polymerase (Ampliqon, VWR) and 1 μL of purified DNA or PCR product. A set of the most common, in-house, used laboratory isolates (3D7 and HB3) as well as P. falciparum negative controls were included in the set-up and the amplified PCR products were analysed by agarose gel electrophoresis.

The assays were performed in 10 μl containing 10x KEY buffer, 500 nM of each primer/probe, 800 μM dNTPs, 0.5 U TEMPase Hot Start DNA Polymerase (VWR) and 2 ng DNA on the CFX96 Touch Real-Time PCR Detection System (Bio-Rad). Thermal cycling conditions comprised 1 cycle of 14′40″ at 95 °C (activation Hot Start Polymerase and denaturation DNA) followed by 40 cycles of 20″ at 95 °C (denaturation DNA) and 40″ at the assay specific combined annealing/elongation/signal detection temperature (Table 1). Specific amplicon generation was checked by evaluating the PCR products using agarose gel electrophoresis. Optimal annealing temperature (Ta) was determined by performing gradient PCR and assessing probe specific signals on Wt/Wt (wild type homozygote), Wt/Vt (heterozygote) and Vt/Vt (variant type homozygote) samples. No template controls (NTC) were included to account for possible contaminations. The Sanger sequenced samples were used for validation and also checked for additional SNVs in the primer and probe binding sites. If present, they were evaluated for their influence on the result. For the assays genotyping SNV 4 and SNV 5 additional primers/probes had to be included for correct genotyping.

The qPCR assays with dual-labeled probes of Bouuaert et al. (2021) were used for genotyping^{54 (link)}. Briefly, for each gDNA sample, genotyping assays were performed in a total volume of 10 µL with 1 × KEY buffer, 250 nM of each primer, 250 nM of each dual-labeled probe, 200 µM of each dNTP, 0.5 U TEMPase Hot Start DNA Polymerase (VWR) and 20 ng gDNA. Primer and probe sequences can be found in^{54 (link)}. The Bio-Rad C1000™ Thermal Cycler with CFX96™ Real-Time System was set at one cycle of 95 °C for 14′40″, followed by 60 cycles of [95 °C for 20″ followed by 40″ of the assay-specific annealing/elongation/signal detection temperature]^{54 (link)}. Data analysis and allelic discrimination plot construction was done with the Bio-Rad CFX Manager 3.1 Software.