Nested PCR || Principle and usage

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Күн бұрын

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.

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@shashpalsidhu2420 Ай бұрын

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.

@Mr.Neelesh_Saha_ 3 жыл бұрын

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@animatedbiologywitharpan 3 жыл бұрын

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@Biocreator 2 жыл бұрын

what if we design specific primer for specific region(that RED region in your video)??

@user-ej6yn8vo2j 9 ай бұрын

am i to understand that the green primer set is depleted relative to red set of primers leading to more specificity of amplicons?

@user-ej6yn8vo2j 9 ай бұрын

are you saying the first primer set, the green pair, acts independent of the 2nd pair? otherwise how are you eliminating nonspecific apmlicons?

@mugdhadeshmukh8037 3 жыл бұрын

Very well explained with diagrams and under 3 mins..

@animatedbiologywitharpan 3 жыл бұрын

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@josa5463 2 жыл бұрын

Your the best sir👌

@zizojaezekeom3565 2 ай бұрын

I still don't understand because i thought a Primer amplifies everything from the binding site to the end of the molecule?

@shashpalsidhu2420 Ай бұрын

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

@niraxlevi9930 Ай бұрын

@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

@marwaahmed3826 2 жыл бұрын

More than thank yoooooooooooooooooooooooooooooooooooooooooooou very much .. simple , to the point and GREAT!

@animatedbiologywitharpan 2 жыл бұрын

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@animatedbiologywitharpan 2 жыл бұрын

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@sonalisahoo3205 3 жыл бұрын

Many many thanks, I appeals to keep it continue.

@animatedbiologywitharpan 3 жыл бұрын

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@sonalisahoo3205 3 жыл бұрын

Sure

@ValmarWings Жыл бұрын

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?

@animatedbiologywitharpan Жыл бұрын

Its used as a mastermix

@shifnaks3898 3 жыл бұрын

Good work sir

@minicat3640 Жыл бұрын

Thank you so much!

@animatedbiologywitharpan Жыл бұрын

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@user-ql8it5xd6v 6 ай бұрын

What ia the rationale of nested PCR?

@priyankanishad7406 Жыл бұрын

Thank sir ji

@wora2965 Жыл бұрын

Do you need to isolate your pcr priduct after first run before you run 2nd pcr? Or do it in same reaction?

@animatedbiologywitharpan Жыл бұрын

Nope

@saraha6980 2 жыл бұрын

Thank you

@animatedbiologywitharpan 2 жыл бұрын

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@piatzin47 10 ай бұрын

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...

@basantapant198 7 ай бұрын

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

@hariprasath3955 6 ай бұрын

Will the two sets of primers have slightly different annealing temperatures?

@amnakhalid2525 6 ай бұрын

Yes both two sets of primers will have diff annealing temperatures according to their nucleotide seq and gene compositions @@hariprasath3955

@anushacu8816 Жыл бұрын

Amazing... explained within 3 min.. better than somu who would take 10min

@animatedbiologywitharpan Жыл бұрын

Please support my channel via super thanks. Your small contribution like 1$ means a lot for us. The super thanks option is present at the lower right corner of any video, a heart sign with $ in it.

@dumindaamila9489 2 жыл бұрын

Great

@animatedbiologywitharpan 2 жыл бұрын

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@misbahhussain1021 2 ай бұрын

What not we use 2nd set of primers first ?

@niraxlevi9930 Ай бұрын

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

@knownymusmusic3268 2 жыл бұрын

Why don't we use secondary primer(specific primer) in the first place?

@rebeccas4801 8 ай бұрын

Same question 😅

@user-ql8it5xd6v 6 ай бұрын

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.

@subratamaity8028 3 жыл бұрын

please make a video on tailed PCR. thank u.

@animatedbiologywitharpan 3 жыл бұрын

Sure I will upload a video regarding that

@karabikalita6321 3 жыл бұрын

Some minutes and the topic is well understood... Thank you❤

@animatedbiologywitharpan 3 жыл бұрын

Please share my channel link with your friends and help me to reach big audience

@animatedbiologywitharpan 3 жыл бұрын

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@karabikalita6321 3 жыл бұрын

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@karabikalita6321 3 жыл бұрын

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@animatedbiologywitharpan 3 жыл бұрын

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@kiplimosimon1429 4 ай бұрын

Excellent

@animatedbiologywitharpan 4 ай бұрын

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@animatedbiologywitharpan 4 ай бұрын

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@afiyatahseenkhan 2 жыл бұрын

Wow

@animatedbiologywitharpan 2 жыл бұрын

Please share my channel link with your friends and help me to reach big audience . Dont forget to check out my entire pcr playlist

@mahammustafa6067 Жыл бұрын

Make a video on loop mediated pcr

@animatedbiologywitharpan Жыл бұрын

I will try

@user-ks9xw4ci7o 6 ай бұрын

هناك شرح بلعربي.

@animatedbiologywitharpan 6 ай бұрын

Could you please help me by sharing my contents with your friends group/ college group. I put huge efforts in making these videos but unfortunately not a lot of people are watching this.