Introduction to Real-Time PCR (qPCR) Techniques

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Background

Using a DNA template, at least one pair of unique primers, deoxyribonucleotides, an effective buffer solution and a thermostable DNA polymerase, the same technique is used to amplify small quantities of DNA as in traditional PCRs. In a thermal cycler containing sensors for measuring the fluorescence of the fluorophore after excitation at the appropriate wavelength, a material marked with a fluorophore is applied to this mixture, enabling the generation rate to be measured for one or more particular items. This makes it possible to calculate the generation rate of the amplified product during each PCR period. The data thus created can be analysed in several samples by computer software to measure relative gene expression (or copy number of the mRNA). In order to assess the occurrence and abundance of a given DNA sequence in these samples, quantitative PCR can also be extended to the identification and quantification of DNA in samples. This calculation is made after each amplification step, and this is why this process is called real-time PCR (i.e. immediate or simultaneous PCR). In the case of RNA quantitation, the template is complementary DNA (cDNA), obtained by ribonucleic acid ( RNA) reverse transcription. The tool used in this instance is quantitative RT-PCR or Q-RT-PCR.


What is qPCR?

  • It is a method used to track in real-time the progress of a PCR reaction.
  • Around the same time, a relatively small quantity (DNA, cDNA or RNA) of the PCR product can be quantified.
  • It is dependent on the identification, as the reaction continues, of the fluorescence emitted by a reporter molecule that increases.
  • It is often referred to as a quantitative polymerase chain reaction (qPCR), a polymerase chain reaction (PCR)-based laboratory molecular biology technique.
  • qPCR is an efficient technique that enables DNA sequences to be multiplied exponentially.
  • A PCR answer requires a pair of primers that are complementary to the interesting series. DNA polymerase is expanded by primers.
  • The copies generated after the expansion, called amplicons are re-amplified with the same primers, resulting in the DNA molecules being exponentially amplified.

However, gel electrophoresis is used after amplification to analyse the amplified PCR products and this takes time with traditional PCR; thus before continuing with the post-PCR analysis, the reaction must stop. PCR Real-Time overcomes this problem.

The word "real-time" means that, in contrast to the traditional PCR system, it will track the progress of the amplification while the operation is going on, where monitoring is only available after the process is finished.


Principle of Real-Time PCR

In real-time PCR, this same concept of amplification of PCR is employed. But the mechanism is tracked in "real-time" instead of looking at bands on a gel at the end of the reaction. The reaction is inserted into a real-time PCR system that uses a monitor or detector to watch the reaction occur.


Although several different approaches are used to chart the progress of a PCR reaction, they all share one thing in common. They all compare the DNA amplification to the fluorescence generation that can clearly be observed during each PCR loop with a camera. Therefore, as the number of gene copies increases during the reaction, the fluorescence increases, signalling the reaction’s success.


MARKER USED IN REAL-TIME PCR

In Real-Time PCR, there are several different markers used, but the most common ones include:

  1. Taqman probe.
  2. SYBR Green.


1.    Taqman Probe

It is a hydrolysis probe attached to the 3 ' end of the oligonucleotide that carries a reporter dye, often fluorescein (FAM) at its 5 ' end and a tetramethylrhodamine (TAMRA) quencher.

The probe remains coiled on itself under normal conditions, bringing the fluorescence dye close to the quencher, which inhibits or quenches the dye's fluorescent signal.

The Taq polymerase oligonucleotide has a homologous region of the target gene and hence binds to the sample DNA when the target sequence is present in the mixture.

As the Taq polymerase starts to synthesise new DNA strands in the extension stage, the degradation of the probe triggers the operation of the 5' end nuclease and the fluorescein is separated from the quencher, resulting in the production of the fluorescence signal.

As this mechanism proceeds, the number of signal molecules increases in each step, producing the fluorescence rise that is positively related to the target amplification.


2.    SYBR Green

This is a dye that emits a conspicuous fluorescent light as it binds non-specifically to the minor groove of DNA.

It is also possible to use other fluorescent dyes such as Ethidium Bromide or Acridine Orange, but due to its greater signal strength, SYBR Green is best used.

SYBR Green is more common than the Taqman Probe as it can provide information on each amplification period as well as the melting temperature that is not collected from the Taqman probe.

Its drawback is the lack of precision as compared to Taqman Probe.


APPLICATIONS OF QUANTITATIVE PCR:

The real-time qPCR is the ocean with numerous applications in various fields, from gene quantification to gene expression. It is also used in food industries, microbial recognition and disease detection, in addition to gene expression research.

ü  Disease diagnosis

ü  Microbial load testing

ü  GMOs detection

ü  microRNA analysis

ü  Cancer detection

ü  Identification and quantification of circulating nucleic acid

ü Detection of pathogenic SNPs


By: Duaa Mughal

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