I'm working on malaria DNA extraction.
In my lab, we have DNA sampled extracted from patient's whole blood 3 years ago. This sample can be amplified by our primers (designed to be specific to 18s rRNA gene and other gene), so I use it as a positive control.
Recently, I used QIAamp DNA Blood kit (Qiagen) to extract malarial DNA from malaria-infected patients who had high % parasitemia, also including the previously PCR-positive sample. I followed manufacturer's protocol with 200 µl of heparinized whole blood (acute phase within 14 days after admission to clinic). Yield of DNA appears to be good 30-40 ng/ul per each 100 ul elution. My positive control always gave positive PCR results. Unfortunately, extracted DNA from new 13 samples and a previously PCR-positive sample gave negative PCR results.
Possible causes that I am considering are:
- incomplete malarial cell lysis during DNA extraction
- the presence of inhibitor(s)
- low malaria DNA compared to human DNA
What I have tried:
- adding more DNA template for PCR reaction
- using prolonged lysis time and increased temperature
I would like to know what factor I have to adjust?
Thank you for your kind response.
Hi, I was facing similar problem while working with a different organism, sometimes you need to modify the actual protocol of kit according to your needs (I also used Qiagen DNA extraction kit). I made changes at three steps and it worked for me:
1- Try increasing the amount of proteinase k to ensure proper lysis.
2- For incubation at lysis step, increase incubation time but incubate samples at room temperature or at 56 degree centigrade for 3 hours.
3- Perform double elution (first elution with short time incubation and second one with increased incubation period at specified temperature) to ensure maximum yield.
Also, make sure that your whole blood samples are EDTA treated.
If that still does not work for you, check out this alternative method for DNA extraction.
Article Purification of genomic DNA from human whole blood by isopro...
I hope it will help solving your problem.
Hi, i'm also working on malaria.
In general, the parasitemia of P. vivax is lower than P. falciparum.
If your PCR is Nested PCR for malaria species identification.
In my experience with 18S rRNA, incresed the DNA template from 1 uL to 2 uL for Nest-1 PCR is work well for me. Most of Nest-1 PCR product can not visulized on the gel until Nest-2 PCR. I used Nest-1 PCR product about 4 uL as a template for the next Nest-2.
-From your DNA concentrations are resonable. However, the 260/280 ratio of DNA should be 1.6-1.8.
-Type of anticoagulant: from the manufacturer, citrate, heparin, or EDTA blood can be used with Qiagen.
-Make sure about protease activity: from the manufacturer, dissolved protease is stable for up to 12 months when stored at 2–8°C.
In my opinion, you are facing up to PCR problem not the DNA extraction.
Hope this answer can give an idea to solve your problem.
Hi, Iqra Kashif and Natpasit Chaianantakul , Thank you very much for your answers. I will try a bit more.
Hi You can use the boiling method for DNA extraction Or use more proteinase k Or use more DNA to test PCR
Iqra, you can try this protocol, it works well when we extract DNA from old samples stored in the freezer:
1. Prepare 1% saponin: 1ml 10% saponin + 9ml UP water
2. Add 1ml of red blood cells (collected with EDTA) to a Corning tube and add 1% saponin to at least 3x the sample volume; vortex;
3. Incubate 5 minutes at 37°C;
4. Centrifuge 3500 rpm for 5 minutes;
5. Remove supernatant, leaving 2ml of pellet.
6. Add 3 to 8ml of UP water; vortex;
7. Centrifuge 3500 rpm for 5 minutes;
8. If necessary, repeat steps 5, 6, and 7 until you get a clear supernatant without hemoglobin.
9. Remove supernatant leaving only a 200μl pellet.
10. Use this 200μl pellet to start extraction with Qiagen.
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