Nested PCR

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

nested pcr

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

Quick detection of Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) i...

Quick detection of Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) in chestnut dormant buds by nested PCR.

Bull Entomol Res. 2012 Jan 27;:1-5

Authors: Sartor C, Marinoni DT, Quacchia A, Botta R

Abstract

Dryocosmus kuriphilus Yasumatsu (Hymenoptera: Cynipidae) develops in chestnut buds that remain asymptomatic from oviposition (June-July) until budburst; it is, thus, easily spread by plant material used in propagation. Therefore, it is particularly interesting to identify infested plant batches before their movement. Unfortunately, a non-destructive method for checking buds has not yet been developed, and the only technique available is the screening of a bud sample. The visual investigation is long and requires highly skilled and trained staff. The purpose of this work was to set up an effective and fast method able to identify the presence of first instar larvae of D. kuriphilus in a large number of chestnut buds by PCR. Four primer pairs were designed on nuclear and mitochondrial sequences of a set of seven gall wasp taxa and tested on five different cynipid's DNA. Nested diagnostic PCR was carried out on DNA extracted from samples of 2 g buds simulating four levels of infestation (larvae were added to uninfested buds); 320 bp amplicon of 28S sequence was chosen as a marker to detect one larva out of 2 g buds. The method showed a potential efficiency of 5000 to 15,000 buds per week, depending on bud size.