Nested PCR || Principle and usage
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- Опубліковано 3 сер 2024
- Nested PCR is a modification of PCR that was designed to improve sensitivity and specificity. Nested PCR involves the use of two primer sets and two successive PCR reactions. The first set of primers are designed to anneal to sequences upstream from the second set of primers and are used in an initial PCR reaction.
to clarify the doubts most people have in the comment section, the first set of primers are added along with the dntps ,buffer , taq polymerase first , then after the first round of amplification, the product from the first PCR reaction is used as the template for the second reaction, where the second set of primers are added.
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Very well explained with diagrams and under 3 mins..
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Many many thanks, I appeals to keep it continue.
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More than thank yoooooooooooooooooooooooooooooooooooooooooooou very much .. simple , to the point and GREAT!
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Your the best sir👌
Amazing... explained within 3 min.. better than somu who would take 10min
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Good work sir
what if we design specific primer for specific region(that RED region in your video)??
Perhaps a silly question, but do you combine both sets of primers in a singular master mix? Or do you add the result after a first PCR run with the second primerset seperately for a second run?
Its used as a mastermix
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Excellent video, but I don't really understand why having two sets of primers minimizes the probability that the second set of primers will bind non-specifically to other regions...
So it involves 2 PCR reactions right. The first PCR product is technically a template for the 2nd PCR. That increases specificity. The mastermix that you design should have outer and nested PCR mix. Outer PCR amplifies a larger region and nested 2nd uses first as template targeting a smaller, internal region of the original sequence. Hope this makes sense
Will the two sets of primers have slightly different annealing temperatures?
Yes both two sets of primers will have diff annealing temperatures according to their nucleotide seq and gene compositions @@hariprasath3955
Some minutes and the topic is well understood... Thank you❤
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@@karabikalita6321 Oh that's really nice.....good to hear about that.
am i to understand that the green primer set is depleted relative to red set of primers leading to more specificity of amplicons?
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are you saying the first primer set, the green pair, acts independent of the 2nd pair? otherwise how are you eliminating nonspecific apmlicons?
Thank sir ji
Excellent
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I still don't understand because i thought a Primer amplifies everything from the binding site to the end of the molecule?
Primers are short sequences of nucleotides that bind to a specific site on the DNA, initiating the replication process. They direct the DNA polymerase to start synthesizing new DNA, but they only amplify the region between the primer binding sites, not the entire molecule. Therefore, only the specific segment flanked by the primers is amplified.i hope this helps
@shashpalsidhu2420 i think that i lack imagination 😂 since this probably doesn't happen immediately as the video shows ,they first amplify the first region by many cycles until they obtain it and then use the second set of primers and proceed the same way
Do you need to isolate your pcr priduct after first run before you run 2nd pcr? Or do it in same reaction?
Nope
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please make a video on tailed PCR. thank u.
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What ia the rationale of nested PCR?
Make a video on loop mediated pcr
I will try
Wow
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Why don't we use secondary primer(specific primer) in the first place?
Same question 😅
We do nested PCR when we have a large size of template and we want to amplify a small portion of it. To avoid non specific amplification, we separate our gene of interest from the rest of the sequence and then amplify it on the basis of its specificity.
What not we use 2nd set of primers first ?
Because we have to shorten the region first by amplifying a smaller region that contains the sequence of interest from the bigger sequence so the chances of amplification of our desired sequence is higher don't forget that sometimes we can amplify directly from a chromosome and that quite huge the primers might bind to another region
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