PCR Basics
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- Опубликовано: 9 июл 2024
- 00:00 Introduction to polymerase chain reaction (PCR)
01:04 Primers
02:34 Primers interact with templates
03:07 Three steps of PCR
03:55 Denature
04:55 Anneal
06:15 Extension; DNA polymerase
07:57 Positions of primers define the length of the amplicon
09:42 "Big picture" view of PCR cycling over time
14:18 Uses of PCR in the study of genetics
14:57 Details of a setting up a PCR experiment
20:27 PCR is sensitive to contamination
So the main thing I learned is that you write on transparent glass and then mirror the video along the y-axis to make it look like you're writing on the glass such that it's in the direction that we, the viewer, read :) Cool effect! People should create art like this...
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's the first to explain this in a manner in which I can understand. Thanks....
This was an extremely helpful video. It helped me a lot to get ready for my presentation. Thank you for your guidance
This is the best and clearly defined video on pcr yet!
Best video on PCR on RUclips... you explain it so gracefully and with great enthusiasm. Thank you!
Very celar, delicately designed, great logic flow and beautiful presentation, I enjoyed it so much. I'm very surprised with this quality your videos haven't gone virus so far. Thanks a lot and please keep making them!
Still benefiting from your class sir. The video was very instrumental in reinforcing the basics of PCR. Thank you sir.
This video is awesome you explain everything perfectly thank you for this
Thanks joseph for the brief explanation. it was well to the point Thanks again for the great job well done
Extremely helpful video. Thanks a million
thank you so much! this one critical step that defines the length of the final amplicons (or at least of the vast majority of amplicons) just wouldn't become clear for me before, but now I think I get it.
Thank you. Easy way to understand for the starter.
really helpful this video. thanks mate.
This is the only video I understand about PCR! While I'm only 15 years old.... THX
Amazing! Perfect explanation!
Wonderful video, thanks
Very well organized. Explanation very clear and easy to follow! Thank you for the helpful video!!!!
Thanks for taking the time to give feedback!
Extremely helpful !
Thank you so much for the information you provided.
Great video! Thanks!
Fantastic video!
Brief and precise.
Thank you for this excellent video! Simple and straight to the point. I always struggle a little bit finding the correct wording when I explain PCR in English, this will help me a lot!
Brilliant,outstanding & mind blowing lecture. THANK you so much Sir for making such helpful videos for us.
Thank you Sir. Really helpful.
I wish to have you as my teacher.
Really great lecture.
To teach this my teacher could have taken 1hr in a very boring manner. Thank god found you.
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!
Fantastic Tutorial Sir!
Very nice presentation, and very clearly explained. Thanks for the resource, and you have a new subscriber.
thanks so much for this.
Great video !
Thank you for the useful video!
Excellent video thank you
excellent, thank you
Thank you for the video, its really help me
Excellent - one of the best explanations I have seen on PCR ( I am not a scientist ).
expalined the PCR so clear! great vedio!
You misspelled video 🤔👀
very well explained
Wow nice video, thanks for helping me understand this topic
Thank you so much
Thank you so much for this video! Helped me so much.
This is a much much better video about PCR than some tens other m ostly by non-English speakers which sound much like exam preparation memorization
Amazing
Wow.. nice explaination 👍👏
Great sir
Wow thank you so much this is soooo cleat and helpful every other video is confusing
Thank you sir ,you really helped.
Nice technology sir 💐
First of all I wanna say I LOVE YOU SO MUCH!!!!!!!!!!!!!!!!!! I wish i had seen you a long time ago, i have watched a lot of videos on P CR and other rDNA Technology Methods, but there is a unique thing about the way you explain the material, your gestures, your tune (Vocal) everything is good a learner... Thank you and we wish to see more of these videos coming, Biochemistry, Molecular Biology and other related subjects.. Thank Mr. Joseph Ross
Helpful
Thanks for this video. do you have any video about the precautions to avoid PCR contamination?
I do not have videos specifically about this topic
I had never understand what does these primaz molecules do, what is their fonction. Guess ı know now. Thx
Hi! I am not sure about what if I have my forward primer bonded to the intron side and 2 bp from the start of my exon (coding region) bonded in to the end of my fwd primer. So my question is when I start reading the codons from my exon, I start it including those base pairs included to my forward primer, or I start reading them after the primer?
I am sorry, but I am not able to provide specific responses to detailed questions that are not directly related to the video content.
Great instructable. Thanks. Questions: (1) after denaturing, during annealing, why are primers more likely to bind to the separated DNA strands than the original full length "negative" strand rejoining its complement? (2) does the primer get replicated as well or does the solution have to have a billion of primer segments at the start?
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
I have a doubt : when we use fragmented DNA molecules digested using restriction endonucleases in PCR we get a particular structure of DNA that doesn't fit perfectly into a vector digested using the same restriction endonuclease. Do we use DNA polymerase Midway ligation or do we carry out PCR of the vector(plasmid) as well??
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))
Hello,
I have a question
By Elisa there are many positive igg with negative igm. And negative pcr? What do this mean with clinical symptoms?
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
wondering how he writes backwards so well :I
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 ;)
Regarding the primers, the jargon you always hear is "upstream" and "downstream." So which one of your two primers is the "upstream" one? Is it the one that anneals to the downstream (3') end of the CODING strand of the template? Or is it the one that anneals to the upstream (5') end of the complementary template strand? Maybe you can see why the jargon might be confusing...
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 RUclips 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.
hello .. i do have a question pls .. i understood how the pcr works ( to amplify and generate billion of copies of targeted DNA )but i don't understand how it helps in the analysis ( or detection of antibiotic resistant gene)
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.
really curious that did he write the opposite way
Hi i have a little confusion, when the DNA polymerase starts copying the template it should extend until the end from 5' to 3' and during that would also copy the other part of DNA or flanking part which is outside the target sequence. So how would we going to get the exact target DNA sequences without any other flanking part. Also, how do we know the primer nucleotide sequence for annealing purpose, if we have not studied that particular genes and how do you isolates the target genes or after doing PCR how you read it. Lastly, I understood that primer cant be denatured bcz it single stranded but when cool it linked to template so why not the separated sequences of target DNA template could also rejoin during cooling process. (Sorry if there is some silly question, i'm not a genetics student)
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..
the primer could attach at multiple sections of the original template - right? makes any difference? not really taq would just keep going until it reaches the 3' end - is my understanding correct?
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..
Why on anneal step DNA doesn't bond with the original counterpart, but with the primer? Or it does but the repetition over comes this hiccup?
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 :)
What sort of black board is this ? Are u writing from back or front of the board sir ???
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 Sir, Can we control DNA polymerase synthesis? Like I don't want to synthesis complete strand. For example - @ - 5' CATGCG 3' is what I want. Like, not synthesizing till the end of chromosome.
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.
COLOR OF PET IS red, what stands for pET in genotyping in genetic analyzer?
PET is an acronym for a fluorescent dye that can be covalently added to DNA. For example, www.ncbi.nlm.nih.gov/pubmed/24869927
Could you please explain how PCR is being used in the covid pandemic and is is accurate? Is is correct that all labs would need to use the same cycle threshold so that positive or negative results are consistent no matter where the testing is done? Similar issue with HIV testing in the 80's? People could be positive for HIV in USA and negative in another country simply because of PCR setup was different?
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..
Why rna primer is not used in pcr
Perhaps because it is less stable than DNA
Like
Sir size of primer clear .S.k.tiwari India.
hOW DNA synthesis stops in PCR ?
DNA polymerase stops synthesizing when it runs out of DNA template to copy.
Thank u ☺️
Its amazing what you can do with a little chromosone and some "hot" bacteria!
I'm confused, I thought primers are RNA
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.
Sit primer will have the chance of having t nitrogenous base sir
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?
speed up at 1.25x. youre welcome
1.75x speed
Taq is an Archea not bacteria
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/
did you write all of this backward?!? AH, I see comment - flipping - better way 8-/
Thanks for watching, Bill!
Why am I watching this, I can't even read good.
Who else thought he was really good a writing backward, but its flipped (ring finger). Get video and explanation non the less