I’m please that you found the video useful

Maybe you could adjust the length of one of the primers slightly to match the annealing temperature of the other? The 5’ ends are fixed but the 3’ end could be adjusted without affecting the size of the amplified product? But I guess you still want a GC clamp also so there a bunch of stuff to consider.

I guess if you think of it as a dynamic process, once the primer is bound (annealed) it stands a very good chance of being extended by the polymerase even at the binding temperature, any extension will bind it more strongly since it is getting longer and therefore will probably still be bound at the higher temperatures up to 72deg. I think that makes sense.

The primers are present at a much higher concentration than the template strands of DNA to be amplified. That is, the single stranded primers are in vast excess. So when the complementary strands of DNA are separated there is a good chance that a primer will bind to its target DNA. If this does not occur, on other copies of the template DNA, the two strands may simply zip back together without the primer binding. But as long as some primers bind to their target the PCR (reaction) will start. Successive rounds of thermal cycling become more efficient because their are more copies of the amplified templates to bind to the primers.

Everything far beyond ~24 cycles is NOT consider as a good parameter for results because the chance of a false results are huge (~97% of the time!). Moreover, PCR is used only for scientific purposes only and not for diagnose purposes. . Why WOH says that theses PCRs test must have to do way more than 24 cycles?! Why WOH uses a scientific method instead of a common one? "Maybe" they do that because this pandemic is a plan-demic, even though they say that this sneeze-19-thing is no more dangerous than a normal flu, this damn pandemonium.

The pre-denaturation occurs only once before the thermal cycle repeats (which may occur 30 times or so). Before the thermal cycling begins the DNA can be considered to be all double stranded and if it's human genomic DNA it's a very large molecule (many millions of base pairs in size). So to begin the PCR an extra stage of pre-denaturation is applied - this simply means the sample is heated for longer at the very beginning before the thermal cycling. For all subsequent rounds of thermal cycling only a short denaturation (heating step) is needed to make the DNA single stranded, also since the PCR product is often quite small (a couple of thousand base pairs) it is easily denatured during a cycle.

No, primers make up the beginning (5' end) of the newly synthesized DNA. The last (3') nucleotide in the primer is covalently bonded to the first nucleotide the enzyme adds just after it attaches. If it didn't work like that, the chain reaction would not be possible - the new strands wouldn't have anywhere for more primers to anneal to in the next cycle. Primers get used up during the reaction, and you would typically add an excess of primers so that you get enough of your target DNA at the end of the reaction.

The polymerase has activity over a wide range of temperatures and it can begin extension of the primer even at 60 degree (72 degrees is the optimum). As extension begins the primer gets longer and it binds more strongly overall. Also the primers do bind at lower temperature but some binging may be less specific whereas a higher or optimum temp will reduce binding to similar but different sites where base pairing may otherwise occur. A low annealing temp will give rise to a variety of PCR products which is typically not what people want. Higher annealing temp will result in a greater proportion of the specific PCR product.

@@UWSMarkTemple Thanks a bunch! Makes total sense.

Very much so. Although the PCR technique used to detect coronavirus uses fluorescently tagged primers - so that the PCR product becomes fluorescently tagged - then using a real-time scanner (during each cycle) the build up of the product can be detected during the linear phase of the reaction. This allows the PCR reaction to be applied as a "quantitative test" - for instance does the PCR product reach a threshold within a number of cycles. There are lots of application for this basic PCR reaction.

@@UWSMarkTemple Can you explain how false positive tests happen, why they happen if they do and why would they happen at higher cycles if that's accurate ?

@@chentobrashnar1442 I'm not Mark Temple, but false positives in PCR testing usually come down to contamination. If a tiny droplet from a strongly positive sample, with say 1000+ times the detection limit, gets into another sample, the other sample will come up positive as well. This can happen any number of ways - the technician might have accidentally picked up a little droplet on their glove and it entered the next tube, or aerosols got emitted from their pipette, or they forgot to change pipette tips, etc. Or perhaps the technician him/herself had Covid without realizing it and contaminated the sample. If the primers were badly designed there could also be nonspecific binding to something else that gets amplified instead, although for Covid tests or other regulated medical tests this isn't likely to be an issue.

@@grebulocities8225 Thanks, so your statement is that only contamination can give false positive results. What if the environment in which lot of people traverse in which multiple sick people are contaminating the air, where most people will breath some particles but far less then the load needed to infect person. Wont that be false positive in our need to identified infection person not just statistic ofsomeone who contain corona virus? Especially if the cycle used is very high, and the lab go with only positive test and not with positive on specific cycle?

@@chentobrashnar1442 I'm not certain that there can't be other causes of false positives, just that contamination is by far the most likely cause. It certainly is possible for contamination to occur in an area with infectious people, although the risk is lower with e.g. drive-thru sites. And yes, false positives would increase the number of reported coronavirus cases. That said, false positives are fairly uncommon, likely around 1% of true positives. False negatives can also happen, especially if the reagents used in the PCR went bad or were added in the wrong concentration.

Indeed, in practice you could use one of many formulas to estimate the annealing temp, but in practice you may need to experiment with a range of temperatures to optimise the experiments under laboratory conditions.