In a nested PCR amplification, a set of primers is first used to amplify the DNA of interest. Then, an aliquot from this primary amplification is used to provide template for a second PCR amplification. This second amplification uses either two new internal primers or one primer from the first amplification with a new internal primer. The sensitivity of most nested amplification procedures is extremely high. Reamplification with the second set of internal primers also serves to verify the specifity of the first-round product, due to the fact that non-specific products from the first amplification are unlikely to act as targets for the nested set of primers in the second amplification. In addition, the transfer of reaction products from the first reaction effectively serves to dilute out inhibitors that might be present in the sample initially. Thus, a conventional nonnested PCR protocol that might have progressed poorly because of suboptimal PCR conditions or inhibition can be salvaged, in many cases, by the use of a nested protocol.
© 2001 Alexander Binder