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.
Method Article
PCR_Sequencing for genotyping SNPs PF3D7_1343700 Kelch protein propeller domain
https://doi.org/10.1038/protex.2013.096
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posted 19 Dec, 2013
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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.
General
Micropipets and tips (10 µL, 200 µL and 1000 µL) 1.5 mL centrifuge tubes
PCR tubes with caps
Disposable gloves
Fine tip marker pens
Nuclease-free water
PCR
10X PCR buffer (MgCl2-free)
MgCl2 (concentration varies)
dNTP (concentration varies)
Taq DNA Polymerase (5U/µL)
Primers for PCR and nested PCR (see Table 1)
Parasite DNA standards: 3D7 at 25 pg/µL
Agarose gel electrophoresis
6X Xylene cyanol dye
Ethidium bromide (10 mg/mL)
Agarose
1X TBE (Tris/Borate/EDTA) buffer
50 bp or 100 bp or 200 bp size standard with xylene cyanol dye added or ready-to-use
SmartLadder MW-1700-10 (Eurogentec)
Parafilm
Thermocycler
Gel electrophoresis apparatus including chamber and power pack
Microwave to melt agarose
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.
3.1. 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.
No
Table 4 Master Mix calculation for Nested PCR for PF3D7_1343700
Table 5 PCR thermocycling conditions for Nested PCR for PF3D7_1343700
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
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted 19 Dec, 2013
You are reading this latest protocol version