Nested polymerase chain reaction (Nested PCR) is a modification of polymerase chain reaction intended to reduce non-specific binding in products due to the amplification of unexpected primer binding sites.

I understand that there are two steps and one of those steps involves a non-specific primer followed by a specific primer...

But this does not seem to have sense. What is the purpose of running a non-specific PCR just to hit it with a specific primer afterwards?

What do we gain when doing a nested PCR?

Practical example: We are planning on sequencing human skin microbiome. Would the ratios of different bacterial species differ if we would use the nested PCR products for sequencing, as compared to standard PCR?

  • $\begingroup$ If your initial template results in multiple amplicons that cannot be removed by increasing annealing temperature, nested pcr can reduce these amplicons when coupled with a gel extraction. $\endgroup$
    – March Ho
    May 26, 2015 at 22:02
  • $\begingroup$ To extend on @MarchHo comment, nested PCR is used for the amplification of a target when too many non-specific fragments are produced by direct PCR. Usually you would use a nested PCR when 1) the sample is complex (multiple DNA sequences) and your target is present in a very small amount as the target would not be amplified otherwise, or more precisely a direct PCR would produce way too many non-specific fragments. 2) When you have many non-specific fragments as a first PCR amplification enriches your "real target" sequence. $\endgroup$ May 30, 2015 at 8:59
  • $\begingroup$ Thanks cagliari. If you could write it as an answer then I would gladly accept it, because it answers my question $\endgroup$
    – WojciechF
    Jun 1, 2015 at 7:28

2 Answers 2


In the paper by K. Lily Therese, U. Jayanthi & H.N. Madhavan traditional PCR wasn't sensitive enough to allow for an accurate reading on what they were trying to study. By using nPCR they were able to amplify around the gene they were interested in and then amplify just the gene they were interested in for a more accurate and sensitive study. The successive PCR allowed them to amplify their specific gene even if there was only one copy in the sample.

I recall using this procedure in Molecular Genetics Lab, it was used to make sure that our sample was purer.


It may depend on which protocol you looking at, but you can use a specific primer set for the first PCR and another specific primer set for the second PCR.

Even if you use a none specific primer set for the first PCR, it could be easier to amplify your target sequence because your target sequence would be more abundant than the original solution before the first PCR is performed.


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