3) Polymerase Chain Reaction (PCR) - Quantitative PCR (qPCR)
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- Опубликовано: 27 апр 2016
- What is Quantitative PCR (qPCR)?
➜ Real-Time PCR or quantitative PCR (qPCR) is a PCR-based technique that is able to simultaneously amplify and detect changes in the amplicon concentration. Real-time PCR collects data during PCR amplification by utilizing fluorescence signals emitted by either special probes or DNA binding dyes.
For more information on qPCR and for a list of the sources used, please visit:
➜ Knowledge Base: info.abmgood.com/polymerase-c...
**Please note that the labels of 5’ and 3’ are flipped at 4:35 - 5:22. Usually R (reporter) is at the 5' end and Q (quencher) is at the 3' end, and thus the top strand bound by the Taqman Probe should be labelled as 3’ on the left, and 5’ on the right. Vice versa for the bottom strand: this should be labelled as 5’ on the left, and 3’ on the right. Please refer to Figure 3 of our knowledge base: bit.ly/2Xwdybj. We apologize for the confusion and thank our youtube community for pointing out the error.
Thank you to all of the amazing community contributions towards translating this video into other languages including:
➜ Indonesian
(To view the translations, toggle the CC button and then go to Settings ➜ Subtitles/CC to select a language.)
Check out our other video series:
➜ Polymerase Chain Reaction (PCR) - An Introduction: • 1) PCR (Polymerase Cha...
➜ CRISPR Cas9: • CRISPR Cas9 - A Brief ...
➜ Adeno Associated Virus: • 1) Adeno Associated Vi...
➜ Cell Culture: • Cell Culture
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I'm a korean veterinary student, and this video makes me deeply and clearly understand about qPCR
THANK YOU!!!
감사합니다 장준영님. 앞으로 업데이트 되는 비디오에도 많은 관심 부탁드립니다!
Direct, clear and specific explanation of fundamentals. Thank you...
Glad it was helpful! Thanks for watching!
nice video, good to study and remember what you have already learned (but not used for some time)
Thanks for watching :)!
Indeed, a wonderful explanation for all the audience to understand the complete process of RT-PCR👏👌👍
Thank you ABM🙏
Hello Shivakumar,
Thanks so much for the awesome comment! :) We are really glad to hear that you found the video helpful. Please stay tuned for upcoming videos too!
Thanks for this awesome video, probably my second comment on youtube :D and the first was yours :D
Thank you for watching our video and commenting! Glad you enjoyed it - if you want to learn more about the topic, you can check out our knowledge base: goo.gl/bUxwkc
This video is really helpful!! Is there any video comparing those DNA polymerase kinds: Tfl, Pwo, Tth and Pfu with Taq polymerase?
In the video, it is mentioned that a drawback with the intercalating dye method is the binding of non-specific sequences. But it also mentioned that the melting curve analysis enables the user to distinguish the amplified products through plotting fluorescence as a function of temperature. So does the mean we can use the melting curve analysis to differentiate between the binding of specific and non-specific sequences as they should have different Tm?
Hello Kenneth! Thank you for watching and leaving a comment! You are correct that the meltcurve analysis can differentiate the products unless (rare but possible) the non-specific product also has a very similar Tm as the specific product. In fact, people use HRM qPCR (high resolution melt) to differentiate a single point mutation in the amplicon. A special HRM qPCR mastermix and a special analysis software are used in those cases!
this makes the concept so easy to understand, thanks a ton :)
but at 2:30, inverse correlation doesn't mean that its equal to reciprocal of nucleic acid in sample! just a minor correction!
Hi Divyae! Thanks for your comment! It is true that the Ct value is more like a relative measure of the concentration of target in the PCR reaction (and not of nucleic acid in the sample, per se)!
such a great video
Thanks so much for watching! :)
Sublime, tank you.
Thanks for watching!
Learn more microbiology on my website. lostonearthandwords.com/category/learning-centre/microbiology/
Are the labeled 5' to 3' strands flipped around the 5 minute mark?
Hi Ryan! Thanks for pointing this out! The labels of 5’ and 3’ are indeed flipped at 4:35 - 5:22. Usually R (reporter) is at the 5' end and Q (quencher) is at the 3' end, and thus the top strand bound by the Taqman Probe should be labelled as 3’ on the left, and 5’ on the right. Vice versa for the bottom strand: this should be labelled as 5’ on the left, and 3’ on the right. Please refer to Figure 3 of our knowledge base: bit.ly/2Xwdybj. We apologize for the confusion and have added the note to our video description.
Please can you tell me the price of this machine?
I have zero understanding on any of this, and after searching for an hour I finally found a video that helped me out!
A question though: what is a DNA dye and how does it get attached to your sample? So from what I understand a viral DNA sample is taken and PCR is done on it, and now I know that at the end this photodetection is done. But what is the step between the two? How does the viral DNA sample emit light for a machine to detect? Second, I saw in an article that said _a patient had a PCR CT value
Regarding the DNA dye, a DNA dye is a molecule that binds to DNA, like BrightGreen and SYBRGreen. The DNA dye can be included in the PCR buffer and binds to double-stranded DNA (dsDNA) by intercalating between the DNA bases. When the DNA polymerase synthesizes the complementary strand to the template, the DNA dye incorporates into the dsDNA and emits a strong fluorescent signal which can be detected by the thermal cycler. Regarding the study, please refer to the authors' interpretation of results.
Hello can you tell me how accurate fetal gender screening is with this method after 20 weeks if you get a female result?
Hi Kristin, this paper published in JAMA in 2011 reviews validity of cell-free fetal DNA testing (detection of Y chromosome sequences in maternal samples) in correctly identifying fetal sex. From the tests they analyzed, they found as high as a 99% positive predictive value from tests using maternal blood. You can read more about it here: goo.gl/ZJqwgg
Bicocca gang
this music makes me have panic attack
If only the background music wasn’t there 😭😭😭😭
Hi Shaheen, thanks for checking out our video and for your feedback! We are definitely trying to find a better balance with the background music. If it really distracts you, you can also try watching the video on silent and turn on the closed captioning for subtitles.