Nested PCR – Nested Polymerase Chain Reaction An Overview

Nested PCR Meaning: Nested PCR full form is a Nested polymerase chain reaction, it’s a modification of polymerase chain reaction intended to reduce non-specific binding in products because of the amplification of surprising/unexpected primer binding sites.

What Is Nested PCR

In the nested PCR (Nested Polymerase Chain Reaction), the specificity of the PCR reaction is enhanced by reducing the non-specific binding with the help of the two sets of primers.
The specificity is the main purpose of any of the PCR reaction. Each PCR modifications are mean to increase the specificity as well as the sensitivity of the reaction.
Because of this, modification in the native PCR technique is always required to achieve the best results.
Nested PCR is an easy modification of conventional PCR which actually increases the specificity of any reaction. The nested PCR is the best option in the microbial identification and 16s RNA analysis.

Two sets of primers are mostly used to achieve high sensitivity in the nested PCR. Therefore, here both primers have totally different and unique properties.

• The first set of primers binds outside of our target DNA and amplifies larger fragments, this set of primer is referred to as an outer primer.
• Another set of primer binds specifically at the target site and in the second round of amplification, it amplifies only the target DNA, this set of primer is named as an inner primer.

Furthermore, in the nested PCR, our template Deoxyribonucleic Acid (DNA) is the primary binding site for the outer set of primers whereas the amplicon of the first set of the PCR (Nested Polymerase Chain Reaction) is the site for binding for the inner set of primers.

Interestingly, the technique doesn’t need any additional reagent, chemical, or instrumentation besides conventional PCR reactions. Only one extra single set of primers is sufficient.
Furthermore, the common drawback with the single set of primer or conventional PCR in the early activation of Taq DNA polymerase, primer-dimer, and also the non-specific bindings of primer to the template DNA.

Role Of Primers

Nested PCR (polymerase chain reaction) involves two sets of primers, that used in two successive runs of polymerase chain reaction, and the 2nd set intended to amplify a secondary target within the 1st run product. Furthermore, it allows amplification for a low number of runs in the 1st round, limiting non-specific products. Therefore, the 2nd nested primer set should only amplify the intended product from the 1st round of amplification and not the non-specific products. Thus, this allows running more total cycles while minimizing non-specific products. Therefore, this is very useful for rare templates or PCR with high background.

Inner Set Of Nested Primers

Even if the non-specific Deoxyribonucleic acid (DNA) sequences can be amplified in the 1st round of polymerase chain reaction (PCR), that non-specific DNA will not be amplified in the 2nd sets of amplification.

The second set of primers is particular to the inner sequence i.e. amplicon of the first round of polymerase chain reaction. It is very unlikely that the inner set of primers binds to other than its specific site because the amplicon from the 1st round of polymerase chain reaction is the template for the 2nd round of amplification.

Outer Set Of Primer

The outer primers are those primers that are upstream to the inner set of primers. furthermore, outer primers are bind to the outside to the flanking region of out target Deoxyribonucleic acid (DNA).
In the 1st round of PCR, It’s possible that this primer will bind to the site other than the target site and amplifies it. therefore, multiple DNA (Deoxyribonucleic acid) bands can be observed and lead to false-positive results.

Polymerase Chain Reaction

Polymerase chain reaction itself is that the process used to amplify deoxyribonucleic acid (DNA) samples, via a temperature-mediated DNA polymerase. Furthermore, the products are often used for sequencing or analysis, and this process is a key part of many genetics analysis laboratories, along with uses in DNA fingerprinting for forensics and alternative human genetic cases. Conventional PCR needs primers complementary to the termini of the target deoxyribonucleic acid (DNA). The amount of product from the PCR will increase with the number of temperature cycles that the reaction is subjected to. A Normally occurring problem is primers binding to incorrect regions of the deoxyribonucleic acid, giving surprising products. Thus, this problem becomes more likely with an increased number of cycles of PCR.

Nested PCR Processes

The target DNA (deoxyribonucleic acid) undergoes the 1st run of a polymerase chain reaction with the 1st set of primers. Furthermore, the selection of uncertainly and similar primer binding sites gives a selection of products, therefore, only one containing the intended sequence.

The product from the 1st reaction undergoes a 2nd run with the second set of primers. It’s very unlikely that any of the unwanted polymerase chain reaction products contain binding sites for both the new primers, furthermore, ensuring the product from the 2nd polymerase chain reaction has little contamination from unwanted products of primer dimers, hairpins, and alternative primer target sequences.

Nested PCR Role In Microbial Identification

The nested qPCR or the nested RT-PCR gives great power to this current technique which might be a useful method for the phylogenetic analysis and identification of different pathogens. Furthermore, the technique has higher sensitivity hence even if the sample contains lower DNA (Deoxyribonucleic acid), it can amplify, which isn’t possible by the conventional PCR technique. additionally, the method is highly specific.
In the nested realtime PCR, the universal primers for 16S and 18S rRNA are used as an outer primer.

Once it amplifies into the PCR machine, the set of species-specific or distinctive sequence primers is used as an inner set of primers.
The 2nd round of PCR or multiplex PCR such as more set of primers for different species, individual PCR, or the target-specific PCR technique can be applied.
Furthermore, the unique sequence primers are specific to one pathogen that amplifies the template DNA if the target sequence is present, therefore, once when the amplification is achieved, then the amount of pathogen present in the sample, which is set quantitatively-ultimately the species of the pathogen can be identified.

By using the universal primer and sequence-specific primer phylogenetic tree for various species of the pathogen can be prepared as well.

Nested PCR Protocal

coworkers and Kamolvarin described the strategy, In the year 1993 how to be used of 2 sets of primers for increasing sensitivity and specificity of the PCR.
The nested PCR reaction is complete into 2 steps, a primary round of amplification with the outer forward and reverse primers. The protocol is as described

Furthermore, when, after the reaction preparation, put the PCR as shown into the below given table.

Furthermore, after the completion of the 1st round of amplification, take the tubes and prepare the reaction for the 2nd round of amplification. After that, you will need to add 1µL – 1µL inner forward primer and inner reverse primer to the PCR tubes of the 1st round of amplification.

Nested PCR Advantages

• It is useful in studies like phylogenetic analysis and genetic polymorphism.
• The main advantage of the present method is that it gives 100% specificity, sensitivity, and accuracy.
• For the not possible templates where the GC content can be high or the prospect of non-specific banding is higher, nested PCR offers the best results.
• It is useful within the amplification of genes with low abundance.
• Further, nested PCR is the best suitable option/choice for carcinoma and viral infection studies.

Yet, due to several limitations, the nested PCR is not the primary choice for many several reactions.

Nested PCR Disadvantages

• The method is long or time-consuming.
• Required a lot of reagents like an extra set of primers and one extra round of agarose gel electrophoresis that means the method is quite costly.
• The chance of contamination is additionally higher.

Conclusion:

Therefore, the nested PCR is one of the gold standard strategies utilized in the identification of pathogens. The combined multiplex-nested PCR technique is used in the study of 16s rRNA and 18s rRNA of HCV and HSV.
Still, the nested PCR is the best option for achieving the specificity, furthermore, it consumes more time. It’s restricted, the technique isn’t appropriate for long-range PCR.

REFERENCES

1. PCR Wiki: Nested polymerase chain reaction
2. Chain Reaction Wikipedia: Wiki PCR
3. Wikipedia primer: PCR Wikipedia
4. Elizabeth van Pelt-Verkuil; Alex van Belkum; John P. Hays (14 March 2008).
5. Article Reference Also Taken From Genetic Education
6. Article Reference Taken From BMC Infectious Diseases
7. “Springer Science & Business Media. pp. 190–. ISBN”