Recombinant taq dna polymerase

DNA was isolated from saliva samples using Oragene collection kits (DNA Genotek Inc., Ontario, Canada) according to the manufacturer's instructions. Approximately 10–40 ng of template was included in each PCR amplification, reactions contained 0.2 mM each deoxynucleotide, 0.2 μM each oligonucleotide, 0.05 U/μl recombinant Taq DNA polymerase with its 1x reaction buffer (NH₄)₂SO₄ (Thermo Fisher Scientific, Pittsburg, PA), and 8% DAT1QuickExtract buffer V1.0 (Epicentre Biotechnologies, Madison, WI) in addition to PCR-specific optimizations. The DAT1 amplification contained 1.5 mM MgCl₂, 0.6 M betaine, and the oligonucleotides DAT1F 5′-TGTGGTGTAGGGAACGGCCTGAG and DAT1R 5′-CTTCCTGGAGGTCACGGCTCAAGG (Shinohara et al., 2004 (link)). Amplification conditions were the following: 95°C 4 min; 35x 94°C 30 s, 65°C 1 min, 72°C 30 s; 72°C 3 min. Amplified products were size separated on a 2% agarose gel (GenePure LE, BioExpress, Kaysville, UT) and visualized using ethidium bromide. Genotype was grouped by 10/10 (homozygous for the 480 bp 10-repeat product), 10/9 (carriers of one 480 bp product), and 9/9 (homozygous for the 440 bp 9-repeat product). Three participants were excluded from data analysis because they carried rare variants of the DAT1 gene. Numbers and frequencies of alleles and genotypes are presented in Table 1.

The expressions of cell-free miRNAs (i.e., miR-223, miR-155, miR-181b, and miR-126) were quantified by miRNA-specific Universal ProbeLibrary (UPL) probe-based stem-loop RT-qPCR method as we previously described [23 (link),50 (link)]. Briefly, this quantification technique included two steps: (1) miRNAs were transcribed into cDNA via reverse transcription using miRNA-specific stem-loop RT primer (500 nM, Integrated DNA Technologies, Leuven, Belgium) and TaqMan^™ MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) and (2) miRNA quantification was performed by RT-qPCR using designed universal reverse primer (100 μM, Sigma-Aldrich, St. Louis, MO, USA), miRNA-specific forward primer (100 μM, Integrated DNA Technologies), and UPL probe #21 (10 μM, Roche Diagnostics) with recombinant Taq DNA polymerase (5 U/μL, Thermo Scientific, Vilnius, Lithuania) and dNTPs (2.5 mM, Thermo Fisher Scientific). All the measurements were run in triplicate on a QuantStudio^™ 12K Flex qPCR instrument (Applied Biosystems). For normalization, the exogenous ‘spike-in’ control cel-miR-39 was measured in all the samples with the same method as above. Primers and qPCR assays were designed by the software developed by Czimmerer et al. [51 (link)], and oligonucleotides that were used in this study are listed in Supplementary Table S1.

For parasite transfection, schizonts purified from an overnight culture of PbDiCre parasites were transfected with 5–10 μg of linearized plasmid by electroporation using the AMAXA Nucleofector device (Lonza, program U033), as previously described [47 (link)], and immediately injected intravenously into the tail vein of Swiss mice. For selection of resistant transgenic parasites, pyrimethamine (35 mg/L) and 5-flurocytosine (0.5 mg/ml) were added to the drinking water and administered to mice, one day after transfection. Transfected parasites were sorted by flow cytometry on a FACSAria II (Becton-Dickinson), as described [44 (link)], and cloned by limiting dilutions and injections into mice. The parasitaemia was monitored daily by flow cytometry and the mice sacrificed at a parasitaemia of 2–3%. The mice were bled and the infected blood collected for preparation of frozen stocks (1:1 ratio of fresh blood mixed with 10% Glycerol in Alsever’s solution) and isolation of parasites for genomic DNA extraction, using the DNA Easy Blood and Tissue Kit (Qiagen), according to the manufacturer’s instructions. Specific PCR primers were designed to check for wild-type and recombined loci and are listed in S2 Table. Genotyping PCR reactions were carried out using Recombinant Taq DNA Polymerase (5U/μl from Thermo Scientific) and standard PCR cycling conditions.