In the nested PCR, the specificity of the PCR reaction is enhanced by reducing the non-specific binding with the help of the two sets of primer.

The specificity is the main aim of any of the PCR reaction. Every PCR modifications are mean to increase the specificity as well as the sensitivity of the reaction. Because of this, modification in the native PCR technique is always required to achieve best results.

Two sets of primers are used to achieve high sensitivity in the nested PCR. Here both primers have different and unique properties.

The first set of primer binds outside of our target DNA and amplifies larger fragment, this set of primer is referred to as an outer primer.

Another set of primer binds specifically at the target site and in the second round of amplification, it amplifies only the target DNA, this set of primer is referred to as an inner primer.

Here, in the nested PCR, our template DNA is the primary binding site for the outer set of primers while the amplicon of the first set of the PCR is the site for binding for the inner set of primers.

Interestingly,  the technique does not require any additional reagent, chemical or instrumentation besides conventional PCR reactions. Only one extra single set of primer is sufficient.