Authors: Didier Menard & Frederic Ariey
This procedure is designed to genotype point mutations on chromosome 13 (PF3D7_1343700) in Kelch protein propeller domain of Plasmodium falciparum, identified in a study by Ariey et al, 2013.
This procedure is intended for use in molecular studies of DNA extracted from dried blood spots or whole blood samples for genotyping of P. falciparum infections. It describes the genotyping procedure for SNPs detection in Kelch protein propeller domain of Plasmodium falciparum (PF3D7_1343700). Full gene sequences are given in Appendix A and SNPs already observed are given in Appendix B. This procedure is applicable for well-equipped laboratories with staff familiar with PCR and sequencing.
Agarose gel electrophoresis
1.1. Prepare Primary PCR Master Mixes in a 1.5 mL centrifuge tube according to the volumes calculated using Table 2. Include enough reactions for DNA controls (3D7) and negative (no template) controls.
1.2. Label PCR tubes and add 20 µl Primary Master Mix to each tube.
1.3. Add 5 µl of template DNA to each tube. Seal and run PCR in thermocycler according to the conditions listed in Table 3.
2.1. Prepare nested PCR Master Mixes in a 1.5 mL centrifuge tube according to the volumes calculated using Table 4.
2.2. Label PCR tubes and add 45 µl Secondary Master Mix to each tube.
2.3. Add 5 µl of Primary PCR product to each tube. Seal and run PCR in thermocycler according to the conditions listed in Table 5.
2.4. Run an agarose gel of Nested PCR product to ensure amplification has been successful (See Section 3).
NOTE: PCR product may be stored at 4 °C for up to 1 week or at – 20 °C or -80 °C for long-term storage.
3.Agarose gel electrophoresis
3.1. Make a 2% agarose gel: Dissolve 2 grams of agarose and 100 mL of 1X TBE in the microwave. Cool, then add 4 µL Ethidium bromide and gently swirl to mix. Pour into assembled gel tray with comb(s) and leave at room temperature for 30 minutes to set.
3.2. Load the gel: Place the gel in gel apparatus and fill to line with 1X TBE. Place 2 µL dots of xylene cyanol per sample on Parafilm. Carefully pipet 10 µL Nested PCR product to each dot of dye. Add 4-5 µL of size standard.
3.3. Run gel at 100-150 volts for 60 minutes and view using a UV transilluminator.
Expected size: 849 bp (Figure 1).
Send 40 µl of N1 PCR products for sequencing, according to the company’s instructions.
Table 1: Primary and secondary forward (F) and reverse (R) PCR primers
Table 2: Master Mix calculation for Primary and Secondary PCR for PF3D7_1343700
Table 3: PCR thermocycling conditions for Primary PCR for PF3D7_1343700
Table 4: Master Mix calculation for Nested PCR for PF3D7_1343700
Table 5: PCR thermocycling conditions for Nested PCR for PF3D7_1343700
Figure 1: PCR products for Nested PCR for PF3D7_1343700
S1: 3D7, S2-S16: tested samples, Neg: PCR negative controls Image source: Didier Ménard, Institut Pasteur du Cambodge.
Figure 2: P. falciparum 3D7 protein coding gene on Pf3D713v3 from 1,724,817 to 1,726,997 (Chromosome: 13)
2181 bp sequences flanking candidate marker SNPs from 3D7 complete genome are given. Positions of primary primers (yellow) and secondary primers (green) are shown.
Figure 3: Polymorphisms observed in the K13-propeller domain
Figure 4: SNPs in PF3D7_1343700 Kelch protein propeller domain already observed
SNPs in PF3D7_1343700 Kelch protein propeller domain already observed
Didier Menard, Malaria Molecular Epidemiology Unit
Frederic Ariey, Unité d’Immunologie Moléculaire des Parasites, Institut Pasteur, Paris, France
Correspondence to: Didier Menard ([email protected])
Frederic Ariey ([email protected])
Source: Protocol Exchange (2013) doi:10.1038/protex.2013.096. Originally published online 19 December 2013.