Nested and Heminested PCR

Nested and heminested PCR are designed to increase the sensitivity of PCR by directly reamplifying the product from a primary PCR with a second PCR. Nested PCR uses two sets of amplification primers and two separate rounds of PCR (Haqqi et al., 1988; Schmidt et al., 1996). The second (nested) set of primers anneal to a sequence internal to the region flanked by the first set. In heminested PCR, the second round of PCR uses one of the first-round primers and one new, internal primer. The amplicon from the second round of PCR is shorter than that of the first (Fig. 11.3). The advantage of nested PCR is increased sensitivity and specificity of the reaction, because the internal primers anneal only if

Target DNA

First primer pair M--

Second primer pair

Second primer pair

Figure 11.3. Nested PCR.

the amplicon has the corresponding, expected sequence. Disadvantages of nested PCR include extra time and cost associated with two rounds of PCR and the increased risk of contamination incurred during transfer of first-round amplification products to a second tube. The physical separation of amplification mixtures with wax or oil (Whelen et al., 1995) and designing the second primer set with a higher annealing temperature are two variations used to reduce the potential for contamination.

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