What is the principle of Nested PCR?

Nested PCR is a method used to minimize nonspecific amplification of DNA templates. Nested PCR involves carrying out two successive amplification reactions, each utilizing a different set of primers. The product generated from the first amplification reaction serves as the starting material for the second PCR, which utilizes oligonucleotides positioned relative to the first primer pair. The first set of primers (referred to as outer primers) amplifies a larger segment of the gene of interest during the initial PCR round. In the second round of amplification, a different set of primers (known as inner or nested primers) specifically target a smaller region within the previously amplified segment. This approach with two sets of primers allows for increased cycling, thus enhancing the sensitivity of the PCR. Additionally, the occurrence of nonspecific binding is minimized. This is because the second PCR reaction ensures that the amplicons from the first reaction predominantly contain the target sequence and its neighboring sequences.