Multiplex 2: crt, mdr1 (1034, 1042, 1246)

Primers

Round 1 PCR primers were designed using Geneious Prime R9 to target larger fragments that surrounded the codons of interest per marker. The forward and reverse primers for pfmdr1.1.2 and pfmdr1.1.3 and the forward primer for pfcrt from (Nag et al. 2017) were used as the round 2 PCR primers, and a novel pfcrt reverse primer was developed using Geneious Prime R9.

Table 4. Details about primers used in Multiplex 2 of the ALARMcoding PCR genotyping protocol
Primer Chromosome Sequence (5' to 3') Reference Nucleotide positiona
Multidrug resistance gene 1 (pfmdr1: Pf3D7_0523000)
mdr1-out-F2 5 CCCAGGTGTTTTATCTGCACA NA 960544-960564
mdr1-out-R2 5 AGATGCAATTCTGTGGGCAA NA 962011-961992
mdr1-in-F2 5 TTTGTCCAATTGTTGCAGCTGTATTAACTTT 2 960672-960702
mdr1-in-R2 5 TGCATTTTCTGAATCTCCTTTTAAGGACATT 2 961159-961129
mdr1-in-F3 5 GGTAAAGTTGATATTAAAGATGTAAATTTCC 2 961259-961289
mdr1-in-R3 5 TGGTCCAACATTTGTATCATATTTATTTGG 2 961810-961781
Chloroquine resistance transporter (pfcrt: PF3D7_070900)
crt-out-F 7 CAAGCAAAAATGACGAGCGT NA 403259-403278
crt-out-R 7 CCAGTAGTTCTTGTAAGACCTATGA NA 404035-404011
crt-in-F 7 TGGCTCACGTTTAGGTGGAGGTTCTTG 2 403491-403517
crt-in-R 7 CATACAAATAAAGTTGTGAGTTTCGGATGTTAC NA 403704-403672
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
mdr1-2-out-F 0.2 μM 0.50 4.00
mdr1-2-out-R 0.2 μM 0.50 4.00
crt-out-F 0.2 μM 0.50 4.00
crt-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.

For both pfmdr1 markers (mdr1-2/mdr1-3), the simplex PCR was performed in separate reactions for each inner primer pair with a total volume of 25μL, with a master mix that contained 1x Buffer, 500 uM dNTPs mix, 5 mM MgCl2, 300 nM per primer (F/R), 1.3 units of GoTaq Flexi Polymerase and 1 μL of PCR product from the first round.

Reagent Final Concentration Volume x1 (μL) Volume x8 (μL)
Water 10.99 87.92
5X Buffer 1x 5.00 40.00
MgCl2 (25mM) 5 mM 5.00 40.00
dNTP 0.5 μM 1.25 10.00
mdr1-2-in-F 0.3 μM 0.75 6.00
mdr1-2-in-R 0.3 μM 0.75 6.00
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.

For both pfmdr1 markers (mdr1-2/mdr1-3), the simplex PCR was performed in separate reactions for each inner primer pair with a total volume of 25μL, with a master mix that contained 1x Buffer, 500 uM dNTPs mix, 5 mM MgCl2, 300 nM per primer (F/R), 1.3 units of GoTaq Flexi Polymerase and 1 μL of PCR product from the first round.

Reagent Final Concentration Volume x1 (μL) Volume x8 (μL)
Water 10.99 87.92
5X Buffer 1x 5.00 40.00
MgCl2 (25mM) 5 mM 5.00 40.00
dNTP 0.5 μM 1.25 10.00
mdr1-3-in-F 0.3 μM 0.75 6.00
mdr1-3-in-R 0.3 μM 0.75 6.00
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.

For pfcrt, the master mix contained 1x Buffer, 500 uM dNTPs mix, 5 mM MgCl2, 200 nM per primer (F/R), 1.3 units of GoTaq Flexi Polymerase and 1 μL of PCR product from the first round.

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
crt-in-F 0.2 μM 0.50 4.00
crt-in-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
    • 62°C/2 min
    • 72/2 min;
  • 72°C/10 min
  • Resting at 4°C.

Final Sequences

Table 5. Details about sequences genotyped in Multiplex 2 of the ALARMcoding protocol
Marker Length PCR_Round Codons
Multidrug resistance gene 1 (pfmdr1: Pf3D7_0523000)
mdr1-2 Round 1 Product 1468 1
mdr1-2 488 2 1034, 1042
mdr1-3 552 2 1246
Chloroquine resistance transporter (pfcrt: PF3D7_070900)
crt Round 1 Product 777 1
crt 214 2 72, 73, 74, 75, 76
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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.