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PCR - Polymerase Chain Reaction Simplified
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- เผยแพร่เมื่อ 14 ส.ค. 2024
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Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies of that particular DNA segment. PCR is now a common and often indispensable technique used in medical laboratory and clinical laboratory research for a broad variety of applications including biomedical research and criminal forensics. PCR was developed by Kary Mullis in 1983 while he was an employee of the Cetus Corporation. He was awarded the Nobel Prize in Chemistry in 1993 (along with Michael Smith) for his work in developing the method.
Typically, PCR consists of a series of 20-40 repeated temperature changes, called thermal cycles, with each cycle commonly consisting of two or three discrete temperature steps. The cycling is often preceded by a single temperature step at a very high temperature (90 °C (194 °F), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.
I've literally watched so many videos on this topic but this is the one that really made it stick. This video helped me so much for multiple reasons, with the first being the neat layout of the video. The second reason is relating the names of stuff to how it plays a role in the whole process, and even going as far as to explain "historical" context. Last but not least, the fact that you related the entire process to a real-life application made it a lot easier to understand how it all fits together, and it made me love it and genuinely want to understand it rather than just study it. Major thanks to you
We have similar things watch this👇
th-cam.com/video/vrG1hmwVQaU/w-d-xo.html
Truly simplified.. One of the best TH-cam channel for learners like me
Watch our latest Video on RT-PCR
th-cam.com/video/7j8STZQWFi4/w-d-xo.html&feature=emb_logo
@@MonaHansen
Agreed
@@MEDSimplified aa
Here are some notes too.
th-cam.com/video/boBaoIHGL6c/w-d-xo.html___Polymerase Chain Reaction
HIV has an RNA genome, not DNA as you state at 6'06" of the video. For HIV, you'd first have to do reverse transcription to produce cDNA before you can do a PCR
Yes
But when this hiv virus enters the human cell, it starts to create dna for making further copies of themselves.
The video is incorrect when it states "the blood of the patient contains the virus, and in the virus is its genetic material, the DNA". Rather, it should state, "the virus's genetic material which is a single stranded RNA molecule, is reverse transcribed within the cell it has infected, and the resulting DNA then integrates into the host cell chromosome".
Useful observation @Alexander Howell
@@alexandrahowell3142 but when hiv virus is already in macrophages then it's dna due to reverse transcription is already integrated in the macrophage cell and further in the T helper cells
The viral RNA is converted to DNA in macrophages.
So we can say that we will directly get the DNA integrated in the cell?🤔
I must confess that by far this is one of the best youtube channels I've come across.Its really #MEDSIMPLIFIED.Bless you.
Thankk you for your awesome comments 😀
Thank you! Automation Engineer here trying to move to the next level of understanding the things that I do.
th-cam.com/video/7j8STZQWFi4/w-d-xo.html&feature=emb_logo
In my book temperatures mentioned in denaturation is 94 degrees Celsius and for 1 minute.
In primer Annealing, temperature is 54 degrees Celsius and time duration is 2 minutes.
Truly simplified
I cant understand anymore and This topics are made me boaring
But now it is very amazing
Thanks a lot
Thank you so much for explaining this topic in such an easy and understanding way!!
So simplified and easy to understand. Brilliant Explanation. Thank you ❤
One of many problems in governments using PCR for mass testing of the coronavirus - why is there no standardized number of cycles to be run to determine a positive or negative test for RNA snippets? It is as if business or political interests choose the results they want first, and then choose the number of cycles to give that result. Say it ain't so Joe.
Oh c'mon man...straight Malarkey! Take that mess to another comment section and quit spreading the nonsense and causing doubt. You don't like per results then go the antigen route.
Diagnosis? The inventor, Kary Mullis said the PCR was of no use for diagnosis.
You are confusing end time PCR with RT PCR. The latter is used for covid testing.
@@danielm.1521 Get a life! What an ignorant response. th-cam.com/video/VW5bNd-D0jw/w-d-xo.html
Look at the Indonesian PCR business..
Really easily understandable ,very informative
Thanks a lot and I'll be back to learn more about this. Now I know why Kary Mullis called PCR a manufacturing process.
I m in love with ur voice it is soo calming soo soothy... Watching video of other teachers on you tube give me headache as their voice is soo noisy but listening to u is really relaxing... And ur presentation is superbb
ThankS to DR Rorpopor Herbal on TH-cam who helps me cured my PCR God bless you DR Rorpopor Herbal his herbal medication is very infective and active
Thank you very much for this video.It is very well explained ,simply and easy to remember
All med simplified videos are grt & nice
Great job, Very helpful, good voice. Thank you
Thanks for the appreciation •°
You really have an amazing voice, the person who is drawing does a good job too.
Very simple explanation of a complex topic👍👍
Thank u so so much. Was of great help.
Thanks a lot sir.
One of the best TH-cam channel for Students like me
Your video is really relevant today for the COVID - 19 pandemic. It helps people understand the testing process of identifying a person that has COVID - 19
You mean understanding that the test doesn't work? Even it's inventor said it should ever be used to test for viruses. No wonder it's 80% inaccurate, a coin toss is more accurate.
@@sportysbusinessThose are facts, thanks for the reminder. Kary Mullis said that you can find whatever you want, just increased the cycles. He also said never to be use to diagnosed just for lab research.
Also contains E.O. In UK under a FIOA disclosure it was found that has EO( carcinogenic agent), way above safe limits. We cant put trust in any man, just Abba.
Thank you sir for trully clearing my all doubt of this topic.
This is so helpful for my report. ❤️ Thanks a lot.
So about the Process of PCR; Denaturation, is when the sample is heated, breaking the hydrogens bonds that holds the double-strand DNA together, resulting the separation of the double-stranded DNA; in Annealing, the sample is finally cooled down, allowing the primers to bind with the individual strands of DNA; in Extension, the primers produce more nucleotides of the short sequence of the primer and once a new DNA is completely formed, the double-strand DNA will also undergo the same steps repeatedly by 20 to 30 times, resulting the millions of copies of DNA.
Correct me if I'm wrong.
Great video!
Just wanted to note that you've mentioned two different temperatures for denaturation, 96 degrees at 4:50 and 72 degrees the rest of the video, which one is correct?
Sarah it is always 96 degrees for denaturation and 72 degrees for extension (a.k.a. elongation). He probably got confused during recording.
thank you for this..i needed a simple and accurate definition n functioning of pcr..for an interview..thank you
Thank you MEDSimplified! You have always helped me in every biochem exam! Thank you for making our student lives easier! Bless you!
One min down in the video already subscribed🙌🏾, amazing explanation thank you!
You are blessing for us sir You explanation is so simplified, 🤧🤧really really helpful , keep making 💜💜
Thanku 🙃
You. Are . Saving. My. Semester. Thank god for you!!!
A worthwhile video for all beginning biology students.
Thank you so much!
jazakAllah hu khaira
Wow thank you for this video!! Very well explained and presented. Super useful.
How is the primer decided? I mean, is the base pair sequence same in all primers? What decides which primer to use?
Thank you ,, it is really really simple and clear explanation ^^
Thank you 😄🤍
Really MED is simplified
My understanding is that the UK is using around 45 cycles for detecting sars-cov-2 which sounds way over the top given this video.
This is end time PCR...which is not used for covid testing
Can you explain why Kary Mullis the inventor of the PCR test stated over and over that the PCR test should not be used to diagnose viruses?
@John Bindini , thank you.
@jorge pearl
Nobody has isolated and/or purified COVID-19. If you believe otherwise then post your *definitive* evidence here.
@@shellysistla9037 - Hi, I'm a complete layman so please excuse any ignorance on my part.
On the subject of the Covid virus, what medical conclusions would you draw from PCR positives where the cycle threshold has been set high - 40 cycles or more - when the video indicates less cycles should be performed (Professor Carl Heneghan has indicated 25 as appropriate) and the test subject is clearly asymptomatic?
Is it correct to refer to such a person as a "case" and is there any realistic possibility of them being infectious?
Thanks. Best regards 👍
@John Bindini I understand that pcr shouldn't be used to diagnose illness. But why do you want people to kiss your ass?
@steven atkinson Bingo!
Thanks so much for this vedio...i easily understood the PCR
Thank you so much
Is this best to detect abdominal tuberculosis
hye, thank you so must for making this topic super easy. my English is not so good so please don,t mind. you are amazing. i am seventeen years old and i want to be a great doctor just like you. you are my top inspiration. thanks again
Thank you so much for the appreciation
Hello. I'm from FLORIDA, USA.
Your English is not bad at all. I understood you perfectly. I wish you much luck in your medical career as a Doctor. I'm sure you will do great things in this field.
Very useful information and well defined...one thing mistake is here an example of Virus HIV containing DNA...🧬
one of the best youtube channels God bless you
Hi, I'd like to request clarification regarding one of the points covered in the video. You say there will be 2 copies after the first cycle, then 4 after the second, and 16 after the third, then 32, 64, 128, and 512. Unless I'm missing something, or misunderstood (which is entirely possible), I think the 3rd cycle should yield 8 copies, not 16. If I'm right, then all of the subsequent values are wrong as well. Additionally, 512 would follow 256, not 128. Otherwise, it's a very helpful video, and I appreciate the work you put into it. Thanks!
You are right .I was searching for this comment so as to confirm that I wasn't wrong
Thanks to clear my doubt
Super Clear voice & best explanation of PCR.Thumbs up ⚘
Sir I have one question
Primer gets bind to the specific site of DNA n then the process starts but what about the other site where primer did not bind..?
That site doesn't need to be amplified
PCR test is not meant to be used clinically for diagnosis..once used it will work as pandemic machine,as in case of HIV nd covid19.
It can diagnose presence of the virus, but not quantity of virus in the sample. This is self-evident if you watch and understand the video.
@@ElectricityTaster I believe what he's saying is that detecting the presence of a single DNA strand is not the same as diagnosing a person with a disease. The ongoing casedemic is an example of the misuse of PCR technology. If I've established immunity and am not courageous, ”PCR only” diagnosis will produce a false positive 100% of the time. Your video is educational about the basics of PCR but misleading when you mention HIV. Maybe elaborating on the importance of knowing where the DNA strand being replicated comes from and the building DNA from RNA.
Thank you-exactly my point.
@Donald Netanyahu Thank you. I seemed to have jumped down the right rabbit hole when I came across your comment. Just subscribed to your channel. 👍😁
Your voice is awesome!
God gifted!
U r not making it simple....u r simply making others mind simple....about a particular topic,...osm work...must appreciate..your work ..all D best...,🤘🤘
Thank you so much DR RORPOPOR HERBAL on TH-cam you saved my life from this deadly PCR virus, I got cured within 14daysly herpesly herpes
Thank you, 👍 it's really superb explanation
Your idea on flashcard is excellent
Thank you this is the best channle for remember biology 🙏❤️👍💯
Please make a video on Recombinant DNA technology
4:37 steps
Thanks
I don't understand some the stuff. Could someone please help me understand?
So if this process only copies a certain part of the DNA, then why are you showing the whole DNA structure again in the copies produced? (the blue parts on your graphics, where only the green part of the DNA was replicated).
Second, what if by mistake the exact same short DNA sequence you want to replicated was already present in the original sample, and it is not necessarily due to an HIV infection (to use your example)?
Third, how do you then analyse these results? How does that machine work, meaning how does a human operate it? And how do you interpret that result?
I'm asking because an article I'm reading mentions that _a patient's PCR value was
Hi. I hope that I can help with answer your questions.
1) I think the TH-camr did that for a visual representation. Their is a method for amplifying the entire genome called "Whole Genome Amplification," (WGA for short), but sadly, I do not know much about that. But, back to your main point here - PCR amplifies a small strand (about 20 - 40 bp) of DNA.
2) In this scenario, you would see amplification, mainly due to the fact that you have the DNA somewhere in your patient's body. To avoid this, many scientists/doctors will make sure that the DNA that needs to be sequenced is unique to the infection that you are searching for. If we take the case of HIV - we scientist have already decoded the entire DNA of the viron, hence, we are able to know for certain what part of the DNA is unique to HIV, and HIV alone. That means that if the DNA sequence for HIV is found in the patient, we can say with a reasonable certanty, that the patient WILL have HIV, and this is not a fluke.
3) Depending on the size of the fragment that you amplified, you can use gel electrophoresis or SDS-Page. These two techniques are essentially the same, but the only major difference is when to use them. We use gel electrophoresis when using large amounts of DNA - usually >50 bp, while SDS-Page will be less than 50 bp. What they essentially do is seperate the DNA fragments based on their size and charge. This is useful to us in say forensics or if their is doubt as to who the parent is. To explain quickly, if we have DNA from a child, and we want to determine the father, we wil take DNA from the suspected father, and run the test. About half of the fragments in the gel will align with the father, the other half aligning with the mother. I understand that this is very brief, but I hope that it gives you an idea on a possible use of PCR.
Another technique for PCR analysis is called "Sanger sequencing." This is essentially where each individual nucleotide is read, and and a visual diagram is shown. From this, we can see the DNA directly. In this case, if we go back to your HIV scenario, we could amplify the HIV specific DNA, and then we would sequence it using this method.
I don't know what you mean by your statement. I know its been a few months, but if you still have any difficulties/have the article, maybe you can send it to me, and I can have a quick read.
Thank goodness i found you
Thanks for the video! However, what about the rate of amplification? Other sources state this is 1-2-4-8-16-32 etc, in this video the step of 4 copies next results in 16 copies. Is it wrong or not?
Thank you it's really simple to understand.
It's so simple and realatable!
The MOST INFORMATIVE AND SIMPLIFIED VIDEO EVER ON PCR! Thanks a lot!
I don’t understand how the multiplication of the DNA shows if you have a certain decease or not? This is what I mostly want to know and it’s still not clear.
Would like to get a video from regarding agarose gel electrophoresis, sturtevant's obsevation, Morgan's linkage theory, etc..
In my book its written that in danaturation template is heated to 94 degrees and you said 72 degrees
Thank you so much, I really appreciated the easy explanation.
Very well explained.. Please share a vedio of Rtpcr procedure for Covid-19..
This is an eye opening explanation. Thanks
Best For Non Medicals😍😍
Good job guys
Salamat pangsagot ko sa biotech 🥰
Well come
96 degrees is the temperature at which DNA denatures, not 72. Once denatured aat 96, annealing of primers to strands takes place at 56 dgerees and then Taq Polymerase comes into action at 72.
The temperature "90-95°C" in denaturing, not 72°C,
This video is literally amazing 😍😍
Grest method of teaching easily understand and learn it thanks a lot sir 😊
Thank you so much. I was searching an easy video. It helped me a lot.
Sir
HIV is a retrovirus having RNA as genetic material but you said that HIV having DNA as genetic material.
Kindly correct it.
If they never isolated Sars-cov 2 to use how primer, how and why are they using PCR test to determinate if a person tests positive or negative for an infectious disease that apparently is never been isolated before?
Exactly. Dr. Sam Bailey's TH-cam asks the same question.
Another question ... why is there no standardized number of cycles to be run to determine a positive or negative test for RNA snippets? It is as if business or political interests choose the results they want first, and then choose the number of cycles to give that result.
Wow what a teaching style ❤❤❤
Does PCR actually "diagnosis" infections? I thought it could tell you whether a sample has viral DNA, but only that. It can't tell you whether someone is "infectious", correct? For that, you'd need to take a "positive" sample and put into a cell culture to see if the virus infected those cells. If that happens, the person is both "positive" for virus and "infectious". Correct? If the cells do not become infected, then the person is not infectious. It's really a two-step process as _part_ of a full clinical diagnosis of illness. Simply having "the virus" tells you very little, yes?
I would recommend you state that the temperatures are measured in C. Everywhere else this video is pretty good as I have an exam on Friday.
Thank you for this video, it really help me a lot
Quite helpful... Thank you for making this video...
The test was invented in the 1980s by Kary Mullis who won the Nobel prize for chemistry as a result.
However, he said it was for research purposes only and should never be used for diagnosis.
Mullis died last year but there is still video of him talking about the test and saying
"If you do it well you can find almost anything in anybody". Deaths from COVID went down to zero in
August and have not risen by very much since. The small rise could just be due to false positive tests.
If the government had not decided it must "test, test, test" it would not have found any justification for
restricting freedom.
Thank you very much for explaining pcv in simple way
Excellent presentation
Thank you so much sir 😊😊
Thank you!
Bro i am studying 9th and we are having cultural event. I am going to explain this topic. CLASS 9..😎
Thanks a million
This was so helpful!
Well done!
Great video. But the temperature for each step could be cross checked. Plus one copy will make 2 then 4 then 8, not 16 as stated in the video. Another thing that you mentioned was that after the first cycle there is one parenteral DNA and one newly formed..it should be that there are 2 copies each with one parental strand and one newly synthesized strand..
Thank you very clear explanation..now i can understand
Good for learning in easy way but many questions can still arises here
Very helpful
Thank you.. Informative
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@@MEDSimplified actually your voice tone! I like it very much! All videos! Are simplified! Am passed my biochem exam because of you thank you brother. I know. Videoscribe is very costly!
What an editing video that's amazing keep it up
Thank you very much❤❤❤
Thank you so much DR RORPOPOR HERBAL on TH-cam you saved my life from this deadly PCR virus, I got cured within 14daysly herpesly herpes