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I have some understanding of how PCR testing works. What I have always been wondering: how can we be sure that a primer reacts with the targeted gene(s) regardless of where¹ the genes are inside a sample?

¹: Specifically with "where" I mean what cells or other barriers they are inside. In other terms my question is: how can we be sure primers reach every possible target?

In more detail: when targeting a gene that is part of the host organism's own genome things might be quite clear, we can have strong assumptions of "where" the gene is. But when targeting genes that are part of organisms that have a parasitic relationship with the host, a very complex system of interactions (mostly called "immune system") should govern the (co)existence of host and parasite. These complex interactions might lead to situations in which the parasitic organism is present in a very special, isolated manner / place in the host. To give one example: micro organisms in the cell walls of macrophages that are about to undergo lysis. Can we be sure that common PCR would detect genes of such micro organisms? Why?

EDIT: as pointed out in the comments the perhaps most correct phrasing is, how do the DNA isolation techniques employed guarantee that primers reach every possible target?

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    $\begingroup$ PCR isn't conducted inside living organisms. The DNA must be isolated first. $\endgroup$ – canadianer Nov 1 '15 at 22:44
  • $\begingroup$ That is correct and was clear to me when posting the question. So my question based on your comment also could be phrased: how do DNA isolation techniques guarantee that all possible targets' DNA is isolated from a sample. I shall update my question with your more precise phrasing, thanks! $\endgroup$ – B M Nov 1 '15 at 22:47
  • $\begingroup$ You will only detect the DNA that you put specific primers in for. You could use random primers, but then you will have a hard time quantifying what you are amplifying. If you are doing Transcriptome analysis in Eukaryotes then you can us a Poly-T primer and Reverse Transcriptase to take mRNA to cDNA. From there you would need to run some form of microarray to detect what it is that you are pulling down. So, no, unless you are putting primers in for specificity for the microorganism you have, you should not get amplification, unless you use random primers. $\endgroup$ – AMR Nov 2 '15 at 1:51
  • $\begingroup$ @canadianer That is false. Colony PCR can allow amplification of bacterial DNA even when the bacteria are added into the PCR mixture while still alive and viable, and the same applies to mammalian cell culture. $\endgroup$ – March Ho Nov 3 '15 at 12:46
  • $\begingroup$ @MarchHo Nowhere in that description do I see PCR conducted inside living organisms. $\endgroup$ – canadianer Nov 3 '15 at 14:56
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There are many methods of DNA isolation, so it is difficult to make broad statements about all situations. Typically, though, during the DNA isolation protocol, essentially all proteins are denatured and removed, as is RNA, cell membrane components, extracellular matrix, etc. If you are using a high-quality well-validated kit from a reputable supplier, your final product should be just DNA. In your hypothetical scenario where parasites have been engulfed by macrophages (BTW, eukaryotes don't have cell walls), as long as the parasitic DNA hasn't been degraded, it will be present at some level after the purification process. As long as it is above the minimum sensitivity of the PCR reaction, any parasitic genes that are being targeted will be amplified.

During the PCR process, the heating step to 95°C completely denatures and melts all the DNA present, allowing the primers unrestricted access to their complementary sequences, where they can then base-pair during the annealing and extension step.

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  • $\begingroup$ Thanks for answering! Can you explain in more detail how the DNA from a microorganism engulfed in the cell membrane of a macrophage will make it through the isolation process? Why does high-speed centrifugation not discard the macrophage entirely? Can denaturation (or another process) as employed in isolation techniques decompose a macrophage's lipid membrane and the engulfed microorganism's cell wall? $\endgroup$ – B M Nov 2 '15 at 14:17
  • $\begingroup$ @BM there are different kits for different types of samples, so if you're processing samples that have cell walls, make sure you choose the correct reagents. The lysis process is quite harsh, denaturing pretty much everything. Look through some of the information at Qiagen, one of the most highly-regarded companies in this field. The centrifugation steps are after lysis (the other answer is rather confusing in this regard), so you're not losing any cells. $\endgroup$ – MattDMo Nov 2 '15 at 14:39
  • $\begingroup$ @BM Here is a selection guide for what type of kit to choose for purifying genomic DNA. $\endgroup$ – MattDMo Nov 2 '15 at 14:42
  • $\begingroup$ Thanks for the link and your comment. I see that the whole issue is more complex than I thought and boils down to what isolation KIT you use. I still want to know much more precisely how those work however, so I will accept your answer and open a new question with more details. $\endgroup$ – B M Nov 2 '15 at 14:49
  • $\begingroup$ @BM thanks. If you search around on Qiagen's website, and go to the product pages for the various kits, you should be able to find documentation on how they work, and what the different buffers do. $\endgroup$ – MattDMo Nov 2 '15 at 14:59
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The polymerase chain reaction is done on free DNA that has been released from the cell by a variety of chemical or physical methods.

There are no cells involved in PCR as the components you don't want are separated by high-speed centrifugation.

PCR also requires the cycling of heat as to aid in the unwinding of DNA for the binding of primers which also allows for the functionality of TAQ polymerase which is a high heat DNA polymerase isolated from the bacterium Thermus aquaticus.

So unless you're talking about a different reaction PCR does not involve any living organisms including cells. They are removed.

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  • $\begingroup$ Thanks for your post! I have perhaps not phrased my question precisely enough. My question as mentioned in the edit can also be phrased: "can we be sure that the isolation techniques will not discard possible targets, e.g. from macrophages?" Taking your post the answer seems to be: "we can be sure that isolation techniques will discard most relevant targets." Can you elaborate? $\endgroup$ – B M Nov 2 '15 at 0:10
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I think the right answer is "they don't". If you were doing pcr on some sample that you didn't know much about, you would certainly include a positive control, meaning a primer that should amplify something that you think is definitely present in your sample. It you get a band for the positive control, then you know you successfully were able to lyse the cells and nothing else interfered with your pcr.

Most modern techniques on typical samples are quite good, so it's rarely a problem. But generally you would prefer to design your experiment to rely on positive results from pcr rather than negative ones.

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