What are the limitations of nested PCR?

There are also several drawbacks. Nested PCR is more time-consuming than conventional PCR, as it involves two rounds of amplification and additional steps for gel electrophoresis analysis. Also, designing nested PCR primers also requires careful consideration to ensure specificity and avoid primer-dimer formation, which can interfere with amplification. Nested PCR also requires prior knowledge of the target’s sequence. This is because it involves designing primers that specifically anneal to regions within the target sequence.