Nested PCR

What Is Nested PCR?

Nested polymerase chain reaction (Nested PCR) is a kind of polymerase chain reaction which reduce non-specific binding in products with the help of the two sets of primer (Fig. 1). Any additional reagent, chemical or instrumentation besides conventional PCR reactions is not required. Nested PCR uses perform two round of PCR with two sets of primers. The products of second round of PCR are the target gene fragments. It generally applied to animals, such as virus, Treponema pallidum, HIV, tumor genes and so on.

Principle of Nested PCR

In nested PCR, two sets of primers are used to promise the sensitivity in the nested PCR. These two kinds of primers have different properties. The outer primers bind outside of target DNA and amplify larger fragments. The inner primers bind specifically at the target site and amplify only the target DNA in the second round of amplification.

Nested PCR Fig. 1  Set up of outer primers and nested inner primers.

The principle of the nested PCR experiment is to design two pairs of primers based on the DNA template sequence, and use the external primers to perform standard amplification of target DNA for 15-30 cycles. After the first round of amplification, a small portion of the initial amplification product is diluted 100-1000 times and added to the second round of the amplification system as a template, using internal primers (bound to the interior of the first round of PCR products) 30 cycles of amplification. The amplified fragments of the second round of PCR were shorter than the first round. Compared to conventional PCR technique, the use of two sets of primers increases the specificity of the amplification because there are fewer target sequences that are complementary to both sets of primers. If the first amplification produces an incorrect fragment, the probability of paired amplification of the internal primer and the incorrect fragment is extremely low, thereby increasing the specificity and sensitivity of the PCR amplification reaction.

It should be noted that we need to pay attention to the ratio of primers for two rounds of amplification: if the first primer is excessive, the remaining primers will also have a certain yield during the second PCR amplification, which is non-specific for the second round PCR reaction product. In the first round PCR, we should try to find out the lowest amount of primers, increase the number of cycles, and use up the remaining primers in the system.

Nested PCR Fig. 2 Principle of nested PCR.

Advantages and Disadvantages of Nested PCR

Nested PCR is very useful in studies such as phylogenetic analysis and genetic polymorphism. More importantly, it gives 100% accuracy, specificity and sensitivity. For the impossible templates where the GC content might be high or chance of non-specific binding is higher, nested PCR offers the best results. It is also useful in the amplification of genes with the low abundance. Further, nested PCR is the best choice for carcinoma and viral infection studies. However, it has some limitations. This method is time-consuming compared with conventional PCR since it involves two round of PCR. Also, nested PCR requires more reagents such as an extra set of primer and one extra round of agarose gel electrophoresis, which means the method cost more. The possibility of contamination is also higher in nested PCR.

Some Subtypes of Nested PCR

Semi-nested PCR: The principle of semi-nested PCR is the same as that of nested PCR, except that one of the primers used in the second round of PCR reaction is the primer of the first round of PCR. This method of PCR amplification twice with three primers is called semi nested PCR.

Reverse transcription nested PCR (RT-nested PCR): RT-nested PCR is developed on the basis of RT PCR. Based on cDNA obtained through reverse transcription, nested PCR amplification of the target gene is performed. It is the same as simple reverse transcription PCR to detect whether a certain RNA is expressed or its relative expression level, but it is more specific and reliable.

Consensus nested PCR: Also known as consensus primer nested PCR. Degenerate primers are designed based on the more conserved sequences in the same genus. Usually, the first round of PCR primers has more degenerate bases than the second round, and the amplification length is 200-300bp. For certain organisms, it is a simple and easy detection method to obtain the target sequence by using consensus nested PCR amplification, and then to obtain information about unknown organisms by sequencing.


  1. Sharifdini M, Mirhendi H, Ashrafi K, et al. Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction with Parasitological Methods for Detection of Strongyloides stercoralis in Human Fecal Samples[J]. American Journal of Tropical Medicine and Hygiene, 2015, 93(6): 1285-1291.
  2. Massung R F, Slater K, Owens J H, et al. Nested PCR Assay for Detection of Granulocytic Ehrlichiae[J]. Journal of Clinical Microbiology, 1998, 36(4):1090-1095.
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