Understanding PCR
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- čas přidán 30. 06. 2024
- This video explains how a Polymerase Chain Reaction (PCR) works and discusses some of the common issues to think about when designing a PCR experiment.
0:00 Introduction to DNA sequences
5:55 Choosing a region of DNA to amplify
8:13 The Thermal Cycling reaction (denaturation, annealing and extension)
13:15 Understanding each round of the PCR reaction doubles the amount of DNA made
18:07 How to estimate primer annealing temperatures
21:22 Achieving DNA binding specificity
26:50 Working through a Thermal Cycling program - the importance of the annealing step
32:16 The problem of primer dimers
34:30 The use of a GC clamp on the 3' end of a primer - Věda a technologie
Excellent. It’s always easy to tell which teachers do the things they’re teaching and which are just repeating text book knowledge they memorized.
This is a professional here.
I’ve watched a lot of videos on DNA... and this is the first time I heard someone say that the 5’ end is so named because the phosphorus is attached to the 5’ carbon on the sugar at that end.
Absolutely amazing. This was extremely helpful, and got me ridiculously excited for PCR. My teacher had us watch this video and take notes on it on our own, to be able to understand PCR, and I am completely blown away at the spectacularly simple demonstrations, and explanations that allowed me to understand this. Thank you so much!
Absolutely fantastic explainer on the mechanics of PCR and primer design. There needs to be an award for the best scientific explainer videos on CZcams because you should certainly win that contest!
Excellent...no where else i could find such beautiful and in depth explanation
absolutely excellent explanation. you have answered my silent question, I read and watch many web. but here I got my answers. thank you so much! I'm relieved
Dear Prof., This PCR video is wonderful!
This is one of the best and one of the most comprehensive video of PCR I have ever come across. It is so detailed and so very easy to understand. All my doubts were easily cleared in this video! Thank you very much!
Thank You So much for this. My PCR knowledge is now increased with quality information. Your quality and rich animations had accelerated and increased my understanding of PCR TECHNIQUE.
Incredible video! One of the clearest explanations of PCR I have gotten! This was so helpful. Thank you so much!
You sir have a gift to transfer knowledge beautifully. Thank you!
Comprehensive and explicit explanation. Thank you Prof.
Noob in PCR and DNA, I learned a lot watching this video. This video explained to me the basics of PCR, and no i understand how this works. Thank you!
Thank you so much. I'm a forensic student from Brazil and your explanation on this topic was the best i've ever seen.
Wow! This is very detailed. It is just what I have been searching for. Thank you so much!
Great video. Explained in Leighmann's terms, very helpful. Thank You
Thank you for the clear and simple explanation!
This is absolutely the best. Sir, you made it so easy to understand. Thanks a lot.
This is a very credible and authoritative lecturer on matters PCR. Keep up the good work sir 🙏🙏🙏
Such an amazing video. Thank you for clearing all my doubts sir.
Thank you so much for explaining about DNA and PCR.
Excellent among all pcr related video on CZcams.
It is my first time watching your video, and it was very helpful for me. As I am a new learner in my laboratory technique, so I need to watch your video again many times more to understand. I am doing my Ph.D. but I never had any background in molecular biology or any skills of PCR.
Thank you for this amazing presentation.
Thank you so much. Very useful and clear explanation 👌🏾
This is understandable for a layman and still good depth. Thanks for the excellent presentation.
I’m please that you found the video useful
Thank you so much, this is exactly what I was looking for!
excellent organized teaching that explains everything
Really an excellent presentation. Thanks
Amazing explanation. Thank you so much, sensei.
Great explanation ! Makes you wonder if Kary Mullis was from this world when he came up with this , genius .
Simply the best. Thank you very much
thank you so much for the clear explanation
Thank you so much for the most comprehensive presentation of PCR.
Your Information is very helpful, Thank you
BIG THANKS Dr. 🙏
I came back to look for you for my exams soon.... Thanks 🙏
Thank you so much for your lesson.
I like your video! Good demonstration, and thank you!!!
This is amazing explanation Sir
this is fantastic, thank you for this!
Amazing explanation
fantastic detail
Thank you so much! it is a great presentation!!
thank you professor. it was a helpful video. really thank you.
Very thorough and easily understood explanation of the basics. Would you consider a discussion of the sources of PCR bias and how to fight them, including those involved with 16S rRNA based microbiota?
I love this explanation
Thank you so much sir! It was really helpful.
Thank you very much.
Excellent. Excellent. Excellent
Great lesson
Really helpful
Thanks a lot Sir
Thank you very much!
Thank you very much!
Thank you very much!
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Excellent!
Excellent .thank you sir
Brilliant 🙏🏻
Nice video!
great video! thank you so much! :)
Great, thank you!
Excellent
Well explained! Most of the designed primers exhibit more or less secondary structures (HairPin, SelfDimers and HetroDimers). What is the criteria to know the level of acceptance for such secondary structures? If few bases are involved while forming secondary structures, will they also melt away while increasing the temperatures? Like the non specific binding of primers to other non specific but similar sequences on the target sequence?
Thank you
thank you
Can you comment on the wobble nuceotide design aspects?
thanks
Dr. Temple, how should I run PCR if I want to amplify a whole gene? In that case I cannot design the primers with close annealing temperatures. Rather, the primers have to attach at the ends of the intended gene.
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.
@marktemple what is your opinion of using pcr to 40 cycles for detection of cv?
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.
Sir, I want to ask question. Is there any different between 16S rRNA and 16S rDNA? is it different from the templeat before PCR reaction (Use DNA or RNA) thankyou
Could i ask question : why we always focus on annealing temperature more than others any sometimes change it to find the optimal temperature !!
Can you please tell me why the double standers of dna don't rebind through annealing process?
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.
professor i request you to make videos on genetic engineering courses . we will be grateful to you.
A bit confused about the temperature to anneal (say 60 degrees) being lower than the temperature to extend (72 degrees). Wouldn't the primer lose binding at 72 degrees? Or is the 60 degree annealing temperature the lower limit for primer binding and presumably holds bond to template to the 72 degree temperature DNA polymerase needs?
Thank you in advance for the clarification!
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.
sir plz explain again the difference b/w pre denaturation and denaturation
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.
As I understood, after a cycle of reaction , primers are not covalently linked to the newly synthesized target DNA. And therefore, the same pair of primers can anneal again to each new strand. Is it right?
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 sequence of the reverse primer doesn't make sense to me. Are you sure that's the right sequence?
can they find CoV 2 using the PCR?
An interesting question: the most suitable extension temperature for polymerase is 72 degrees. If the annealing temperature for the binding of primer and DNA strand is 60 degrees, will the primer fall off the DNA strand when the temperature increases to extension step? If so, how does the polymerase work?
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.
Are you sure you can amplify a DNA using this technique?
Didnt explain how to decide how long to make the extension time so you know the target region is replicated and nothing more.
👍👍
Is this tool useful with our current dilemma?
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.
I think G C increase 4 degree celcius not 3 °C
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.
Thank you