Nested PCR: Principle and Applications

By Acharya Tankeshwar •  Updated: 06/21/21 •  4 min read

Nested PCR is a modification of PCR designed to increase the sensitivity and specificity of the assay reaction. It involves the use of two primer sets directed against the same target and two successive PCR reactions.

Nested PCR (Image Source: )

The first set of primers is designed to anneal to sequences upstream from the second set of primers, whereas the second set of primers is situated internally or nested with respect to the first set of primers. First set of primers also called “outer primers” amplify a large fragment of the gene which is used as a template in the second round of PCR that targets a smaller region of the amplicon using the second set of primers also known as “inner primers or nested primers.”

The traditional approach to nested PCR was to perform a number of PCR cycles using first set of primers, and then open the reaction vessel and add the second, nested, set of primers to run second PCR cycle. The major problem with this approach is amplicon contamination in the laboratory and a consequential loss of specificity of the assay. To address this issue single-tube nested PCR (STNPCR) reactions have been developed, wherein both sets of primers are added to the initial reaction vessel and an extended PCR is performed.

Amplicons from this PCR assays are visualized by electrophoresing the reaction mixture in 2% ethidium bromide-stained agarose gel along with a molecular weight marker.

Nested Polymerase Chain Reaction (PCR)

Nested PCRs are sometimes necessary to compensate for inefficient first-round PCR due to primer mismatches so, if we can use well-matched primers for first-round PCR nested approach may not be needed in many circumstances.


Nested PCR reduces the nonspecific amplification of the target sequence.  

This is because nested primers will not find priming sites on any primer dimers or nonspecific artefacts generated in the primary PCR. Nested primers will only prime any specific product generated in the primary PCR, thus maintaining PCR specificity.

Applications of Nested PCR

To improve the sensitivity of the assay, nested PCR has been used in many PCR assays. It is particularly useful for suboptimal nucleic acid samples, such as those extracted from formalin-fixed, paraffin-embedded tissue

Nested PCRs have proven valuable for the detection of microorganisms when they are present in very low quantities. For example:

Commercial application

The BioFire FilmArray from bioMérieux is a commercially available system that employs nested, multiplex, and singleplex PCR reactions for the detection of a variety of pathogens.


References and further reading

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. I am working as an Asst. Professor and Microbiologist at Department of Microbiology and Immunology, Patan Academy of Health Sciences, Nepal. If you want me to write about any posts that you found confusing/difficult, please email at

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