What is the Difference Between Conventional Nested and Real-time PCR Assays

The key difference between conventional nested and real-time PCR assays is that conventional PCR is a technique developed to amplify specific sequences of DNA and nested PCR is a modification of conventional PCR that consists of two sequential amplification reactions, while real-time PCR is a variant of conventional PCR that is able to quantify the amplified product.

PCR is a very common scientific technique widely used in research and medicine to detect DNA. PCR tests are used to detect antigens by detecting their DNA or RNA. Generally, viral RNA is present in the body before detecting antibodies or displaying the symptoms of the disease. A PCR test can tell whether or not someone has the virus early on. Currently, PCR is the standard test for detecting COVID-19 disease. There are different types of PCR techniques such as real-time PCR, nested PCR, multiplex PCR, hot start PCR and long-range PCR, etc.

CONTENTS

1. Overview and Key Difference
2. What are Conventional PCR Assays
3. What are Nested PCR Assays
4. What are Real-time PCR Assays
5. Similarities – Conventional Nested and Real-time PCR Assays
6. Conventional vs Nested vs Real-time PCR Assays in Tabular Form
7. Summary – Conventional vs Nested vs Real-time PCR Assays

What are Conventional PCR Assays?

Conventional PCR assay is an in vitro DNA amplification technique routinely performed in molecular biological laboratories. This method enabled the production of thousands to millions of copies of a particular DNA fragment. Kary Mullis introduced this technique in 1980. This technique requires a DNA fragment known as the template in order to make many copies of it. Taq polymerase works as the DNA polymerase enzyme and catalyzes the synthesis of new strands of the template sequence.

Primers in the PCR mixture will work as the starting points for the fragment extensions. All the ingredients that are necessary to make copies of DNA are included in the PCR mixture. PCR reaction is run in a PCR machine, and it should be fed with the correct PCR mixture and the correct PCR program. If the reaction mixture and the program are correct, it will produce the required number of copies of a particular section of DNA from a very small amount of DNA.

There are three major steps involved in a PCR reaction: denaturation, primer annealing, and strand extension. These three steps occur at three different temperatures. PCR buffer maintains the optimal conditions for the Taq polymerase action. These three stages of PCR reaction are repeated to produce the required amount of the PCR product. At each PCR reaction, the number of DNA copies doubles. Hence, exponential amplification can be observed in PCR. PCR product can be resolved using gel electrophoresis since it produces a visible amount of DNA on a gel, and it can be purified for further studies such as sequencing.

PCR is a valuable tool in medical and biological research. Especially in forensic studies, PCR has an immense value since it can amplify DNA for studies from the tiny samples of the criminals and make forensic DNA profiles. PCR is widely used in many areas of molecular biology including, genotyping, gene cloning, mutation detection, DNA sequencing, DNA microarrays, and paternity testing.

What are Nested PCR Assays?

Nested PCR is a type of PCR that reduces the non-specific amplification of DNA. There are two successive PCRs or two sequential amplification reactions in nested PCR assay. During the first amplification reaction, a PCR product is produced. After the first reaction, a second amplification reaction is performed on the PCR product of the first reaction. Therefore, primers in the second reaction mixture bind with the first PCR product and amplify it.

Primer pairs are different in each reaction. The non-specific binding of primers is reduced in nested PCR. Nested PCR assays are useful to increase the sensitivity and/or specificity. However, nested PCR requires knowledge about the interested sequence.

What are Real-time PCR Assays?

Real-time PCR or quantitative PCR (Q PCR) is a modified version of PCR that measures the PCR products quantitatively. Therefore, this technique quantifies the amplification in real-time using a real-time PCR machine. It is also a suitable method for determining the amount of a target sequence or gene present in a sample.

The interesting feature of real-time PCR is that it combines both amplification and true quantification into a single step. Therefore, the need for gel electrophoresis for detection can be eliminated by the real-time PCR technique. The use of fluorescent dyes to label PCR products during the PCR reactions will eventually lead to direct quantification. When the PCR products are accumulated, the fluorescent signals are also accumulated, and they will be measured by the real-time machine. SYBR Green and Taqman are two methods to detect or watch the amplification process of the real-time PCR. Both methods monitor the progress of the amplification process and report the quantity of the product in real-time.

Real-time PCR has a wide variety of applications such as gene expression quantification, microRNA and non-coding RNA analysis, SNP genotyping, detection of copy number variants, detection of rare mutations, detection of genetically modified organisms, and detection of infectious agents.

What are the Similarities Between Conventional Nested and Real-time PCR Assays?

• Nested and real-time PCR assays are modifications of conventional PCR assay.
• All three techniques amplify DNA samples.
• Their products can be used in sequencing or analysis.
• These assays require primers.

What is the Difference Between Conventional Nested and Real-time PCR Assays?

Conventional PCR is a technique developed to amplify specific sequences of DNA. Meanwhile, Nested PCR is a modification of conventional PCR that consists of two sequential amplification reactions, and real-time PCR is a variant of conventional PCR that is able to quantify the amplified product.  So, this is the key difference between conventional nested and real-time PCR assays. Unlike conventional and real-time PCR, nested PCR uses two primer sets. Moreover, there are two successive amplification reactions in nested PCR in order to reduce non-specific amplification. besides, the conventional and real-time PCR assays do not contain two sequential amplification reactions.

The following infographic lists the differences between conventional nested and real-time PCR assays in tabular form for side by side comparison.

Summary – Conventional vs Nested vs Real-time PCR Assays

Conventional PCR is the first technique developed to amplify specific fragments of DNA. Nested PCR and real-time PCR are two variants of conventional PCR. There are two sequential amplification reactions and the use of two primer sets in nested PCR. Real-time PCR is developed to quantify the amplified PCR product. Thus, this summarizes the difference between conventional nested and real-time PCR assays.

Reference:

1. Green MR, Sambrook J. “Nested Polymerase Chain Reaction (PCR).” Cold Spring Harbor Protocols, U.S. National Library of Medicine.
2. “Real-Time Polymerase Chain Reaction.” An Overview | ScienceDirect Topics.

Image Courtesy:

1. “Polymerase chain reaction” By Enzoklop – Own work (CC BY-SA 3.0) via Commons Wikimedia
2. “Nested PCR” By Zephyris at the English-language Wikipedia (CC BY-SA 3.0) via Commons Wikimedia
3. “Real time PCR” By İnformatic – Own work (CC BY-SA 3.0) via Commons Wikimedia