Evaluation of single nucleotide polymorphism associated with resistance to pyrimethamine Pfdhfr (codons 51, 59, 108,164), sulphadoxine -Pfdhps (codons 436, 437, 540, 581, 613), artemisinin-partner drug such as lumefantrine and amodiaquine - Pfmdr (86, 184, 1042, 1246), and chloroquine Pfcrt (72–76) were done using the Qiagen TypeIT master mix. First, the primers were reconstituted and diluted to 10X, from this, 0.7µM and 1X of Qiagen master mix, 3.3 Μl of nuclease free water and 1 µL of gDNA (Supplementary Table 1 ) was used for the HRM drug assay as described earlier [31 (link)] For each drug target, mutant and wild type strains of laboratory cultured adapted parasites and nuclease free water were used as controls.
Type it master mix
Manufactured by Qiagen
Sourced in Germany
Type-it Master Mix is a high-performance, ready-to-use PCR mix for reproducible and sensitive results. It is suitable for a variety of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Rox 500, by Thermo Fisher Scientific (1 mentions)
Abi 3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Genemarker v2, by SoftGenetics (1 mentions)
Genemapper v4, by Thermo Fisher Scientific (1 mentions)
Genescan 500 liz size standard, by Thermo Fisher Scientific (1 mentions)
Abi prism 3730 sequencer, by Thermo Fisher Scientific (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
4 protocols using type it master mix
1
Pyrimethamine, Sulphadoxine, Artemisinin Resistance Evaluation
2
Mitochondrial COI Sequencing Protocol
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At least fifty individuals were chosen from each sample. Mitochondrial COI (mtCOI) sequences were obtained by PCR amplification with the general primer set C1-J-2195 (5′-TTGATTTTTTGGTCATCCAGAAGT-3′ ) and L2-N-3014 (5′-TCCAATGCACTAATCTGCCATATTA ) [26 ]. The PCR was conducted in a final volume of 20μL, 2x Type-it master mix (QIAGEN), 20μmol/L of each primer, and 10 ng of insect DNA extract. A first step of denaturation at 94°C for 5 min was followed by 40 cycles at 94°C for 20sec, 52°C for 30sec, with a final elongation step for 10min at 72°C. Each PCR product was sequenced (Macrogen Inc., Sequencing Service, Korea). DNA sequences produced in this study were identified using the BLASTn algorithm of GenBank (http://www.ncbi.nlm.nih.gov ) and submitted to GenBank. Eighteen other mtCOI sequences were obtained from public sequence databases using the Tax browser (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html ). Multiple sequence alignments were constructed using the CLUSTALW-based alignment tool available in MEGA version 6 [27 (link)] and by manual editing.
3
Microsatellite Genotyping Protocol
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We used fifteen microsatellite loci extensively validated in previous studies and using the same previously published protocols[49 (link),50 (link)]. Primer sequences for amplification and chromosomal locations of the loci can be found in S2 Table . Amplifications were performed with fluorescently labeled forward primers (6-FAM and HEX) using a standard PCR in 13 μl reaction volumes containing approximately 100 ng of genomic DNA, 5 μl of Type-it Master Mix (Qiagen, Germany) and 1 μl each of forward and reverse primers (10 μM starting concentration). PCR products were then multiplexed, combined with size standard (Applied Biosystems ROX500) and highly deionized formamide, and genotyped on an ABI 3730xl DNA Analyzer (Applied Biosystems Inc, USA) at the DNA Analysis Facility on Science Hill at Yale University (http://dna-analysis.yale.edu/ )). Alleles were scored using the program GeneMarker v 2.4.0 (Soft Genetics, State College, PA, USA) with manual editing of the automatically scored peaks.
4
Microsatellite Genotyping of Larval and Adult Samples
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Genomic DNA from all larval (n = 125) and adult (n = 212) tissue samples was isolated with commercial Qiagen DNeasy Blood & Tissue Kits following the instructions of the manufacturer. For genotyping, we selected 15 microsatellites among the set described in Gutiérrez-Rodríguez et al.
(2014). These loci were grouped into three multiplex reactions (Table S2) with Multiplex Manager This article is protected by copyright. All rights reserved.
Microsatellite amplification was implemented by polymerase chain reaction (PCR) in a total volume of 15.2 μL, with 7.5 μL of Type-it Master Mix (Qiagen), 1.2 μL of primer mix, 5.3 μL of RNase-free H 2 O, and 1.2 μL of template DNA.
PCR cycling conditions consisted of an initial denaturation step at 95ºC for 5 minutes, followed by 30 denaturation cycles for 30 seconds at 95 ºC, annealing for 90 seconds at 60ºC and extension for 30 seconds at 72ºC, with a final extension phase of 10 minutes at 60ºC. The quantity and quality of PCR products was verified through electrophoresis in 2% agarose gels, including negative controls in each PCR to detect possible contamination. PCR products were genotyped in an ABI PRISM 3730 sequencer with the GeneScan 500 LIZ size standard (Applied Biosystems). Finally, the multilocus genotype of each individual was assigned based on the size of the amplified fragments using GENEMAPPER v4.0 (Applied Biosystems).
(2014). These loci were grouped into three multiplex reactions (Table S2) with Multiplex Manager This article is protected by copyright. All rights reserved.
Microsatellite amplification was implemented by polymerase chain reaction (PCR) in a total volume of 15.2 μL, with 7.5 μL of Type-it Master Mix (Qiagen), 1.2 μL of primer mix, 5.3 μL of RNase-free H 2 O, and 1.2 μL of template DNA.
PCR cycling conditions consisted of an initial denaturation step at 95ºC for 5 minutes, followed by 30 denaturation cycles for 30 seconds at 95 ºC, annealing for 90 seconds at 60ºC and extension for 30 seconds at 72ºC, with a final extension phase of 10 minutes at 60ºC. The quantity and quality of PCR products was verified through electrophoresis in 2% agarose gels, including negative controls in each PCR to detect possible contamination. PCR products were genotyped in an ABI PRISM 3730 sequencer with the GeneScan 500 LIZ size standard (Applied Biosystems). Finally, the multilocus genotype of each individual was assigned based on the size of the amplified fragments using GENEMAPPER v4.0 (Applied Biosystems).
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