Thanks for noticing! I wish I could take credit for the technology, but it was designed by two different universities simultaneously: it is known as a Lightboard by some and by Learning Glass by others.

He was an amazing professor! He made things easy to understand and make it so you’ll understand it in the long term not just to past the class! I took a couple of his classes and his classes are the only ones I can say that I fully remember every aspect of it! Even tho it’s been 4 years!

Thanks for taking the time to give feedback!

You misspelled video 🤔👀

It is a production "trick" - I write normally, but after the video recording is complete, it is flipped, so I am "backward" (my right side is on the left), but the writing looks normal to the viewer.

mind blown

Ok. Where did you write it? To Glass or something else?

on concrete, yasin kaya

He can write backwards because on the video he is also "left-handed" and wears a wedding ring on the "right" hand ;)

Both questions have the same answer! (Great questions to ask, also!) Primers do not get replicated during PCR, so the process only has as many (as you indicate, millions/billions of) primer molecules as added at the start. It is this extremely high concentration of primers (relative to template DNA molecules) that answers your first question. Once the double-helices are denatured, there are so many more primers than "negative" strands floating around that when the solution cools, one (of the millions of) primer molecule will almost certainly be positioned to base-pair with its complementary target sequence.

@@JosephRoss Thank you for your speedy reply. Much appreciated. Makes a lot of sense. A few related questions, if you are up for it: (Q1) I saw in the NIST database that you can use multiple primers to PCR a specific segment, for example there are 6 different primer pairs to amplify "AATG" STRs from THO on chromosome 11. Now, most of these primers do not end with the same nucleotides, meaning they are cutting at different positions in the strand. The resulting amplified strands must have not only the the primer, the STR alleles (one or two types), but also some extra luggage, basically nucleotides between the primer and the STR. Since all lengths, but the lengths of STRs are known, are these simply subtracted as overhead from the total strand lengths to determine the allele size of the STRs? Or is there a more elaborate process of "cleaning" the strands first, before they go into the "gel" for electrophoresis? (Q2) After denaturing, it appears that primers attach spontaneously to their targets, just before the polymerase enzyme collects free floating nucleotides and "plugs" them into the open strand. How come the relatively long primers (20bp+) attach spontaneously, while the single nucleotides don't without polymerase? Are primers electrically charged? (Q3) Are there situations in which primers do not "find" their targets because the subject DNA has either an indel or an SNP polymorphism in the very segment the primer would otherwise attach to? Thanks again, and if this is getting too much work, I fully understand.

@@ikoknyphausen198 1: I think you have the correct idea. Regardless of which pair of primers is used, the _difference_ in lengths of PCR products is the same. 2: single nucleotides will temporarily hydrogen-bond (base-pair) with other nucleotides, like those found in single-stranded DNA templates. However, only after DNA polymerase connects multiple nucleotides together do they (collectively) have enough hydrogen bonds to stay (almost always) base-paired to the complementary strand of DNA = double-stranded DNA, the double-helix. Thus, a primer (usually 20+ nucleotides) binds to its target long enough for DNA polymerase to start adding nucleotides to the 3' end, while a single nucleotide isn't present long enough for DNA polymerase to initiate replication. 3: Absolutely, polymorphisms/mutations in primer sites on template DNA molecules can prevent primers from binding

Great point - thanks for bringing it up. Usually, geneticists use "upstream" and "downstream" in a slightly different context: relative to the location of a gene (or particularly the transcription start site) on a chromosome. So, in general, "upstream" means "on the 5' end of" (or "before," as we read DNA sequence left to right from the 5' to the 3' end), and "downstream" means "on the 3' end of " or "after." This is just colloquial language: to my knowledge there is no formal definition of these terms. With respect to PCR, my perspective is that geneticists typically refer to the "forward" ("upstream") primer and the "reverse" ("downstream") primer. The forward/upstream primer is written 5'-to-3' and binds to its template strand with the 3' end on the right end of the primer. The reverse/downstream primer, when annealed to its template strand, would have the opposite polarity (with its 3' end on the left side of the primer). It is, indeed, difficult to describe very clearly in writing (and it would be great if images could be attached to KZfaq comments!) I also want to note that, relative to PCR, there is not necessarily a coding strand, as you mentioned, because PCR primers can be designed to amplify any part of a chromosome, including outside of genes - and the coding strand terminology only relates to genes.

I do not have videos specifically about this topic

www.lightboard.info (writing from behind, and then the video is mirrored over the vertical axis so I am backward and the writing is forward - for example, I'm not left-handed…)

Hi Adil. Around 10:31, I begin explaining how DNA polymerase uses one primer to extend (synthesize) the second DNA strand. Yes, you're right, that polymerase will keep synthesizing for as long as it can (until the PCR cycle ends - when the temperature returns to the denaturation temperature of 95°C). At 12:26 and onward in the video, you'll see that, over time, the length of the majority of new template molecules created by the PCR process are defined by the positions of the two primers. So, you are right that every new DNA strand created by PCR from an "original" (chromosome-length) template molecule will be longer than intended, but from the second round of amplification onward (through the ~30th), the length of each new strand is limited by where the primers anneal to the template. The ultimate effect is that, after 2^30 new strands are created, only a tiny fraction of those are the "wrong" (unintended size); most are exactly the same length, dictated by the primer locations. Now, to your second question (which is a very important question to ask, so thanks for that): which came first: the chicken or the egg? Which came first: known DNA sequences to design primers with, or primers used to amplify DNA for sequencing? Fortunately, the answer is pretty mundane: some of the earliest primers were random nucleotide sequences. The concept is that, using short primers of random sequence, there is a finite chance that both primers will randomly anneal close enough together and in the correct orientation on a chromosome to produce a product that could be cloned and sequenced. Once you know some of the actual DNA sequence, then primers with specific sequence can be designed, and off you go sequencing more of the genome. Another approach to genome sequencing relies not on PCR amplification of the DNA to sequence, but rather creating artificial chromosomes containing fragments of the target genome (e.g. fragments of human DNA in bacterial chromosomes) and then relying on bacterial replication (instead of PCR) to create billions of copies of that DNA molecule. Thus, no primers are required. This is how the earliest DNA sequences were obtained.

Thank u so much for such detailed reply..

Unfortunately, because I'm unfamiliar with the specific details of those tests, I cannot comment on their accuracy

@@JosephRoss LMAO! Yet, you just did! Anyone who does any research learns pcr can give 80% false positives, and "were never intended to diagnose anything." according to at least one of Kary Mullis' colleagues. Obviously the question wasn't about how accurately the typical covid test facility performs. You know full well how accurate pcr can be as a covid19 test under ideal conditions. Why not give us your opinion on a best case scenario, instead of a cowardly excuse?

@@kelhawk1 he's afraid.. but he KNOWS it's not designed for diagnosis..

Thank you sir ,you really helped.

I'm sorry, I can't respond to such detailed questions, because it would require a lot more information and time to fully understand the research project (and thus the potential problem(s))

I am sorry, but I am not able to provide specific responses to detailed questions that are not directly related to the video content.

It is this extremely high concentration of primers (relative to template DNA molecules) that answers your question. Once the double-helices are denatured, there are so many more primers than "original counterpart" strands floating around that when the solution cools, one (of the millions of) primer molecule will almost certainly be positioned to base-pair with its complementary target sequence before the other template strand can do so.

@@JosephRoss thank you :)

Each primer can base-pair with any single-stranded DNA (including the original template) anywhere there is a series of "matches" (A with T; C with G) between the primer and template. Once the primer is bound, Taq will synthesize DNA from its 3' end. The main way this influences the quality of PCR analysis is that if both primers randomly happen to bind near each other on the template at multiple locations, then there will be a mixture of PCR products, which usually is not desirable. Usually the desired outcome of PCR is the selective amplification of the one chromosomal region being targeted.

@@JosephRoss Innthe case of sars 2 ..what is the primer... Is it 100 nucleotides in length or less and which section is concentrated on to amplify...

@@kdcruz75 He's a coward.

@@kelhawk1 Dont know what they have to gain? They want slavery. They will get it.. But some of us ..are looking to move the courts ajd human rights commission. If they fail..then it is on record that they r ignoring truth to violate our basoc god goven rights... Then will come universal jurisdcition... ..then there will be no mercy...everyone contributing to this fraud will be rounded up.. Medics, adminsitrators, police..politiciians.. All charged under universal jurisidction in a military tribunal. They will be moved to laboir camps and forced labour..the very camps they think they r building for us..

If you know the DNA sequence of a gene, you can design primers that will amplify part of it. Then, if you have a DNA sample and want to know if that gene is present, PCR will produce a detectable amount of DNA from that gene; if the gene is not present, no PCR products will be created.

That is exactly the benefit of PCR. The 5' ends of the two primers define the ends of the piece of DNA that is synthesized. In reality, you can't use PCR to create a 6-nucleotide product, like you ask about in your question, but for the purposes of understanding, if you wanted to create that product, then in theory you would design two primers (one, 5'-CG-3' would anneal to the 3' end of the sequence you wrote; the second, 5'-CA-3' would anneal to the 3' end of the complementary strand, which you didn't provide in your question). This is the point I make starting at 7:57 in the video.

Joseph Ross Thank you sir.

I'm not an expert in the ELISA technique; you would be better served asking somebody else (sorry!)

Thank you do you know of someone that can assist

PET is an acronym for a fluorescent dye that can be covalently added to DNA. For example, www.ncbi.nlm.nih.gov/pubmed/24869927

Perhaps because it is less stable than DNA

DNA polymerase stops synthesizing when it runs out of DNA template to copy.

Thank u ☺️

I don't understand the point you're trying to make. Can you explain more, please? Also, what time in the video is related to your point?

It is an understandable and common question - you are definitely right to be confused! In the normal process of DNA replication in living cells (in vivo), DNA synthesis must be primed with a primer (a single-stranded length of nucleic acids complementary to the opposite strand). This primer is synthesized in the cell by the enzyme called primase, and it creates RNA, not DNA. So, this would be an RNA primer, and this RNA primer is later removed and replaced with DNA so that the newly synthesized nucleic acid strand is entirely DNA. The difference here is that PCR is an in vitro process: we conduct it "in glass" (in tubes - really, plastic ones these days, but that's a trivial point). For various reasons, we don't use primase to generate primers - we (the researchers) simply provide the in vitro reaction with DNA primers that we've paid companies to synthesize for us.

Thanks for watching, Bill!

I suppose it depends on which taxonomist you ask - and perhaps when. The original description of Thermus aquaticus described it as a bacterium: www.ncbi.nlm.nih.gov/pmc/articles/PMC249935/