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I have previously asked a more general question about how PCR sequencing reaches all possible DNA targets here. I want to ask more specifically now: what evidence is there that common PCR DNA purification kits can isolate DNA from pathogens engulfed in macrophages?

Specifically what such a KIT would have to achieve is to destroy the double lipid cell membrane of a macrophage and destroy the cell walls of engulfed bacteria or fungi. (The latter of which even contain chitin)

I am looking for as specific evidence as possible proving that if one takes a whole blood sample and employs a purification KIT, any microorganism's DNA engulfed in a macrophage should be isolated. Please allow me to say that I am looking for evidence beyond just the manufacturers specifications. I would like to find publications, book chapters or other written statements that specifically show and discuss how isolation techniques work on the barriers I mentioned or even better how they work on intra-cellular micro organisms. Thank you!

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  • $\begingroup$ What do you mean by a "PCR DNA purification kit" ? Can you link to such a kit? $\endgroup$
    – March Ho
    Nov 3, 2015 at 12:42
  • $\begingroup$ Also, colony PCRs routinely work on either colonies of bacteria or isolated mammalian cell culture, so there is no reason bacteria inside mammalian cells won't work. I can't say the same for fungi, since I have not worked with yeast. $\endgroup$
    – March Ho
    Nov 3, 2015 at 12:45
  • $\begingroup$ @March Ho: the link you asked for, qiagen.com/de/shop/sample-technologies/dna-sample-technologies/… $\endgroup$
    – B M
    Nov 3, 2015 at 19:51
  • $\begingroup$ @March Ho: so your reasoning ist that since isolation techniques work on mammalian cells and on bacteria, they should also work on one inside the other? That might be the case but does not follow by pure logic! The kit you use on one (bacteria/mammalian cells) might not work on the other and even if you use the same kit for both it might not isolate the dna of bacteria inside mammalian cells for reasons of: too short exposure time or not enough reactants or competitive binding or unforseen reactions or altered cell wall of intracellular bacteria or... $\endgroup$
    – B M
    Nov 3, 2015 at 20:02
  • $\begingroup$ I didn't use a kit, but merely ran a PCR using the cells instead of purified DNA as the template. $\endgroup$
    – March Ho
    Nov 3, 2015 at 21:16

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For this problem, I would generate the evidence myself taking the following approach:

Apply the DNA extraction kit to the macrophages with pathogens engulfed. As controls, also use the DNA extraction kit on the macrophages alone (negative control), and the pathogen alone (positive control 1). Additionally get purified genomic DNA of the pathogen for a better positive control.

Now, design primers which specifically amplify pathogen DNA. You should test the primers against the independently purified pathogen gDNA to ensure they are working. I would focus on generating 200 bp fragments, so that your primers can be used for qPCR later. Since it seems you are interested in potentially a high throughput sequencing, you may want to target multiple genes to see if there is a difference in efficiency.

Now use your kit on all the samples (Macrophage + pathogen, Pathogen alone, Macrophage alone) and then run PCR on those purified samples. If you detect pathogen DNA from the macrophage + pathogen sample, this would give you strong evidence that the kit is capable of simultaneously purifying both DNA sequences. Otherwise, it would confirm that this co-purification does not work. You can make the technique better by incorporating qPCR to see if the efficiency of extraction is significantly reduced by having the pathogens inside the macrophages.

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  • $\begingroup$ This does not provide an answer to the question. Once you have sufficient reputation you will be able to comment on any post; instead, provide answers that don't require clarification from the asker. - From Review $\endgroup$
    – tyersome
    Sep 10 at 2:51
  • $\begingroup$ @tyersome I am a new user with a Ph.D. in synthetic biology. Can you elaborate on why your feel this does not answer the question? My procedure would help this user to generate the answer to this question, which I feel would be very valuable to a practicing academic. Additionally, this post had no answer for half a decade. My answer provides something versus nothing. Please let me know why this is inappropriate for Biology Stack exchange. $\endgroup$ Sep 10 at 11:08

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