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?
Answer
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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