Multiplex 3: dhfr, dhps

Primers

The round 1 PCR primers were designed using Geneious Prime R9 to target larger fragments that encapsulated the primers described in (Nag et al. 2017) that were used as the round 2 PCR primers.

Table 6. Details about primers used in Multiplex 3 of the ALARMcoding PCR genotyping protocol
Primer Chromosome Sequence (5' to 3') Reference Nucleotide positiona
Dihydropteroate synthase (pfdhps: PF3D7_0810800)
dhps-out-F 8 ACAACACACAGATATAGCATACTTTT NA 549342-549367
dhps-out-R 8 AATAGCTGTAGGAAGCAATTGC NA 550530-550509
dhps-in-F1 8 ACCATCAGATGTTTATATAACAAATATGTG 2 549417-549446
dhps-in-R1 8 CTGGATTATTTGTACAAGCACTAATATCA 2 549909-549881
dhps-in-F2 8 AGAATGTGTTGATAATGATTTAGTTGATAT 2 549845-549874
dhps-in-R2 8 GATATAAAAGTTGATCCTTGTCTTTCCT 2 550365-550338
Dihydrofolate reductase (pfdhfr: PF3D7_0417200)
dhfr-out-F 4 GCCATTTTTGTATTCCCAAATAGC NA 747913-747936
dhfr-out-R 4 TGTCATCATTCTTTAAAGGCATATCA NA 748853-748828
dhfr-in-F 4 ATGATGGAACAAGTCTGCGACGTTTTCGA 2 748088-748116
dhfr-in-R 4 CTAAAAATTCTTGATAAACAACGGAACCTCC 2 748610-748580
Note: Blank references denote that the primer was created by this author. Manuscript in preparation.
1 2 Nag et al., 2017
a Nucleotide position relative ot the 3D7 v3 reference genome

PCR Instructions

Round 1.

The first round PCR was carried out in a total volume of 25 μL with 1 x buffer, 500 uM dNTPs mix (Promega), 5 mM MgCl2, 200 nM of each primer (forward (F) and reverse (R)), 1.3 units of GoTaq Flexi Polymerase (Promega) and only 2 μL of genomic DNA.

Reagent Final Concentration Volume x1 (μL) Volume x8 (μL)
Water 9.49 75.92
5X Buffer 1x 5.00 40.00
MgCl2 (25mM) 5 mM 5.00 40.00
dNTP 0.5 μM 1.25 10.00
K13-out-F 0.2 μM 0.50 4.00
K13-out-R 0.2 μM 0.50 4.00
mdr1-1-out-F 0.2 μM 0.50 4.00
mdr1-1-out-R 0.2 μM 0.50 4.00
G2 Flexi Taq (5U/μL) 1.3 U/μL 0.26 2.08

Cycling conditions:

  • 95°C/2 min
  • 40 cycles of:
    • 95°C/20 sec,
    • 58°C/2 min,
    • 72/2 min;
  • 72°C/10 min
  • Resting at 4°C.

Round 2.

The second round of the nested PCR was carried out in separate reactions containing a single inner fragment primer pair and with a total volume of 25 μL containing 1x Buffer, 500 uM dNTPs mix, 5 mM MgCl2, 200 nM of each primer (F/R), 1.3 units of GoTaq Flexi Polymerase and 1 μL of PCR product from round 1.

Reagent Final Concentration Volume x1 (μL) Volume x8 (μL)
Water 11.49 91.92
5X Buffer 1x 5.00 40.00
MgCl2 (25mM) 5 mM 5.00 40.00
dNTP 0.5 μM 1.25 10.00
dhps-1-in-F 0.2 μM 0.50 4.00
dhps-1-in-R 0.2 μM 0.50 4.00
G2 Flexi Taq (5U/μL) 1.3 U/μL 0.26 2.08
Reagent Final Concentration Volume x1 (μL) Volume x8 (μL)
Water 11.49 91.92
5X Buffer 1x 5.00 40.00
MgCl2 (25mM) 5 mM 5.00 40.00
dNTP 0.5 μM 1.25 10.00
dhps-2-in-F 0.2 μM 0.50 4.00
dhps-2-in-R 0.2 μM 0.50 4.00
G2 Flexi Taq (5U/μL) 1.3 U/μL 0.26 2.08
Reagent Final Concentration Volume x1 (μL) Volume x8 (μL)
Water 11.49 91.92
5X Buffer 1x 5.00 40.00
MgCl2 (25mM) 5 mM 5.00 40.00
dNTP 0.5 μM 1.25 10.00
dhfr-in-F 0.2 μM 0.50 4.00
dhfr-in-R 0.2 μM 0.50 4.00
G2 Flexi Taq (5U/μL) 1.3 U/μL 0.26 2.08

Cycling conditions for each simplex reaction:

  • 95°C/2 min
  • 40 cycles of:
    • 95°C/20 sec
    • 58°C/2 min
    • 72/2 min;
  • 72°C/10 min
  • Resting at 4°C.

Final Sequences

Table 7. Details about sequences genotyped in Multiplex 3 of the ALARMcoding protocol
Marker Length PCR_Round Codons
Dihydropteroate synthase (pfdhps: PF3D7_0810800)
dhps Round 1 Product 1189 1
dhps-1 493 2 431, 436, 437
dhps-2 521 2 540, 581, 613
Dihydrofolate reductase (pfdhfr: PF3D7_0417200)
dhfr Round 1 Product 766 1
dhfr 523 2 16, 50, 51, 59, 108, 164
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References

Nag, Sidsel, Marlene D Dalgaard, Poul-Erik Kofoed, Johan Ursing, Maria P Crespo, Lee O’Brien Andersen, Frank Møller Aarestrup, Ole Lund, and Michael Alifrangis. 2017. “High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology.” Scientific Reports 7 (1): 2398. https://doi.org/10.1038/s41598-017-02724-x PMID - 28546554.