Definition of Nested PCR :
Nested PCR is a variation of the polymerase chain reaction (PCR), in that two pairs (instead of one pair) of PCR primers are used to amplify a fragment.
The first pair of PCR primers amplify a fragment similar to a standard PCR. However, a second pair of primers called nested primers (as they lie / are nested within the first fragment) bind inside the first PCR product fragment to allow amplification of a second PCR product which is shorter than the first one.
The advantage of nested PCR is that if the wrong PCR fragment was amplified, the probability is quite low that the region would be amplified a second time by the second set of primers. Thus, Nested PCR is a very specific PCR amplification.
The Nested PCR Reaction
Nested PCR requires two sets of primers which are used to amplify a specific DNA fragment using two separate runs of PCR. The second pair of primers function to amplify a smaller specific DNA fragment located within the first PCR product.
Nested PCR Reaction Diagram
Steps of the Nested PCR
Step One: The DNA target template is bound by the first set of primers shown in blue. The primers may bind to alternative, similar primer binding sites which give multiple products however only one of these PCR products give the intended sequence (multiple products not shown).
Step Two: PCR products from the first PCR reaction are subjected to a second PCR run however with a second new set of primers shown in red.
As these primers are NESTED within the first PCR product, they make it very unlikely that non-specifically amplified PCR product would contain binding sites for both sets of primers. This nested PCR amplification ensures that the PCR product from the second PCR amplification has little or no contamination from non-specifically amplified PCR products from alternative primer target sequences.
Nested PCR protocol
Nested PCR : Publications
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