Nested PCR
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
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
A nested PCR method for the identification of hepatitis B virus genotype in p... Related Articles
A nested PCR method for the identification of hepatitis B virus genotype in paraffin blocks of formalin-fixed liver biopsies.
Arch Iran Med. 2008 Jul;11(4):455-8
Authors: Geramizadeh B, Kaboli R, Behzad-Behbahani A, Rahsaz M, Azarpira N, Aghdai M, Aytollahi M, Yaghoobi R, Baneehashemee M
Hepatitis B virus is a hepatotropic virus that causes acute and chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma; it is responsible for more than one million deaths annually worldwide despite hepatitis B vaccination. Until now, there are eight known genotypes (A-H).Clinical course of chronic hepatitis B varies according to the genotype of Hepatitis B virus. Liver biopsy becomes necessary to judge the degree of liver lesions and to make the final diagnosis, especially to make the diagnosis for latent liver damage and early-compensated cirrhosis. Genotype is very important for prognostication, but it has not yet been reported on liver tissues. Sometimes, it can be helpful to do genotyping of Hepatitis B virus of the liver tissue; such conditions include research programs, when serum is not available or when serum is negative for Hepatitis B virus DNA. In this study, we wanted to evaluate the feasibility of a simple method for genotyping of liver tissue samples. We performed genotyping of the liver biopsies and intended to find out a simple and reliable method for genotyping in the paraffin- embedded formalin- fixed liver tissue.Genotype D was the only isolated genotype in all the liver biopsies. The tissue genotype was just the same as that found in serum. The procedure was easy and good for large scale studies.Genotyping in the paraffin-embedded formalin-fixed stored liver tissue can be done with the same accuracy of the serum.
PMID: 18588380 [PubMed - in process]
Development and validation of nested-PCR for the diagnosis of clinical and su... Related Articles
Development and validation of nested-PCR for the diagnosis of clinical and subclinical infectious laryngotracheitis.
J Virol Methods. 2008 Jun 25;
Authors: Chacón JL, Ferreira AJ
A standardised nested-PCR method that amplifies a region of the glycoprotein E gene of avian infectious laryngotracheitis virus (ILTV) has been developed for the diagnosis of infection by Gallid herpesvirus 1. The two sets of primers employed produced the expected amplification products of 524bp (external primers) and 219bp (internal primers) in the presence of ILTV DNA, whereas no such amplicons were obtained with other avian respiratory pathogens or with DNA extracted from the cells of uninfected chickens. The identity of the 219bp amplified product was confirmed by DNA sequencing. The standardised nested-PCR method detected ILTV DNA from trachea, lung, conjunctiva and trigeminal ganglia samples from flocks of birds with and without clinical signs, and showed high sensitivity (95.4%) and specificity (93.1%) when compared with the reference test involving virus isolation in specific-pathogen-free chicken embryos. The standardised nested-PCR method described may be used to detect clinical and latent ILTV infections, and will be of significant value for both diagnostic and epidemiological studies.
PMID: 18584884 [PubMed - as supplied by publisher]