Advantages & Limits of Nested PCR vs. Standard PCR

Nested polymerase chain reaction (PCR) is a modification of PCR that was designed to improve sensitivity and specificity of the technique. Nested PCR involves the use of two primer sets and two successive PCRs; an initial PCR generates a product that is used as the template for the second round of amplification. The purpose of nested PCR is to increase assay specificity by re-amplifying the target from a template that was previously enriched. Off-target sequences that may have been amplified non-specifically in the first PCR are therefore not re-amplified in the second round of PCR since they are unlikely to possess the internal priming sites targeted by this subsequent reaction. The use of two pairs of oligonucleotide primers also allows a higher number of cycles to be performed, thereby increasing the sensitivity of the technique.

Fig. 1

Simplified process of nested PCR. An expansion of the initial standard assay, the second step uses the amplified product of the first procedure, resulting in significantly improved specificity, and often improved sensitivity as well. Figure made in BioRender.

The key factor in nested PCR involves the two pairs of primers being targeted to a single locus; the first pair amplifies the target template along with some additional sequence while the second pair anneals to sites within those amplicons to amplify an internal, shorter, sequence. After the first PCR, a second PCR procedure is performed on the produced amplicons with primers that bind to that same locus on the target sequence. Amplicons from nested PCR assays can then be detected in the same manner as conventional PCR. In this way, the first PCR product must be efficiently amplified from the desired sequence to allow the second reaction to generate an appropriate product.

There also exists a sister method to nested PCR, termed semi-nested PCR. Whereas in nested PCR both the forward and backward primers used in the first round of PCR are replaced, in semi-nested PCR only one (or even none) of these primers are replaced prior to the second round of amplification. Additionally, both fully nested and semi-nested approaches can be used with real-time PCR (rt-PCR) technologies.

Nested PCR has been widely used to detect the presence of various viral and bacterial pathogens in the food industry and in a clinical setting with levels of sensitivity higher than that achievable by traditional PCR. Nested PCR is useful especially on suboptimal nucleic acid samples, like those extracted from formalin-fixed, paraffin-embedded tissue. Nested PCR is also increasingly useful when it is necessary to increase the sensitivity or specificity of PCR, for example, when amplifying a particular member of a polymorphic gene family or when amplifying a cDNA copy of an mRNA present at a very low abundance.

Nested PCR is an efficient method to amplify segments of long templates but requires sufficient knowledge of the target sequence for appropriate, effective, primer design. Although this technique is increasingly sensitive, false positives due to contamination or amplification of nonspecific sequences have been a notorious issue. For this reason, stringent precautions and great care must be exercised when performing a nesting PCR experiment.

To minimize carryover, different parts of the process should be physically separated from one another. These contaminants have also been controlled in the literature by using primers designed to anneal at different temperatures, though this requires much higher levels of experimental optimization.