Nested PCR || Principle and usage

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.

[shashpalsidhu]

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_]

I start with thumbs up 👍 before I finish each of your videos !

[mugdhadeshmukh]

Very well explained with diagrams and under 3 mins..

[piatzin]

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...

[zizojaezekeom]

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

[anushacu]

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

[sonalisahoo]

Many many thanks, I appeals to keep it continue.

[Biocreator]

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

[ValmarWings]

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?

[richard-po]

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

[richard-po]

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

[wora]

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

[marwaahmed]

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

[misbahhussain]

What not we use 2nd set of primers first ?

[knownymusmusic]

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

[karabikalita]

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

[subratamaity]

please make a video on tailed PCR. thank u.