Real-Time PCR (qPCR): Principle, Protocol, Fluorescent Markers, Advantages & Applications

Table of Contents

• Introduction to Real-Time PCR (qPCR)
• PCR Terminology
• Principle of Real-Time PCR
• Steps of Real-Time PCR (Protocol)
• Fluorescence Markers used in Real Time PCR
• Advantages of Real-Time PCR
• Applications of Real-Time PCR
• References

Introduction to Real-Time PCR (qPCR)

• Real-Time PCR is a technique used to monitor the progress of a PCR reaction as it occurs in real time.
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• It allows the quantification of a relatively small amount of PCR product, whether DNA, cDNA, or RNA.
• The method relies on detecting fluorescence produced by a reporter molecule, which increases as the reaction proceeds.
• Real-Time PCR is also known as quantitative polymerase chain reaction (qPCR), a molecular biology technique derived from conventional PCR.
• qPCR enables the exponential amplification of DNA sequences.
• A PCR reaction requires a pair of primers that are complementary to the target sequence of interest.
• These primers are extended by DNA polymerase during the reaction.
• The newly formed copies, called amplicons, are repeatedly re-amplified by the same primers, leading to exponential amplification of DNA molecules.
• In conventional PCR, amplified products are analyzed after the reaction using gel electrophoresis, making the process time-consuming because post-PCR analysis can only be done once amplification is complete.
• Real-Time PCR overcomes this limitation by allowing analysis during the amplification process.
• The “real-time” aspect refers to the ability to monitor and measure the amplification progress while the reaction is still ongoing, unlike conventional PCR where results are visible only after the entire reaction has finished.

PCR Terminology

• Polymerase Chain Reaction: PCR
• Reverse Transcription–Polymerase Chain Reaction: RT-PCR
• Real-Time Polymerase Chain Reaction: qPCR
• Combined Reverse Transcription and Real-Time PCR Technique: qRT-PCR

Principle of Real-Time PCR

• Real-Time PCR uses the same amplification principle as conventional PCR, but instead of observing DNA bands on a gel at the end, the entire process is monitored in real time.
• The reaction is run in a real-time PCR machine equipped with a camera or detector that continuously observes the amplification as it happens.
• Multiple techniques exist to track the progress of a PCR reaction, but they all share a central concept: linking DNA amplification to the production of fluorescence.
• As fluorescence is generated during each cycle, the camera detects it, allowing real-time measurement.
• The fluorescence intensity increases as the number of gene copies increases, making it possible to monitor the reaction’s progress cycle by cycle.

Steps of Real-Time PCR (Protocol)

The working procedure can be divided into two steps:

A. Amplification

Denaturation:

• A high-temperature incubation is applied to separate double-stranded DNA into single strands and to loosen secondary structures in single-stranded DNA.
• The highest temperature that the DNA polymerase can tolerate is generally used, typically 95°C.
• The denaturation time may be increased if the template DNA has a high GC content.

Annealing:

• During this phase, complementary sequences are allowed to hybridize.
• An appropriate temperature is selected based on the melting temperature (Tm) of the primers.
• The annealing temperature is usually set 5°C below the primer Tm.

Extension:

• This step occurs optimally at 70–72°C, the temperature at which DNA polymerase shows maximum activity.
• Primer extension happens rapidly, at a rate of up to 100 bases per second.
• In real-time PCR, where amplicons are typically small, the extension step is often combined with the annealing step and carried out at 60°C.

B. Detection

• The detection phase in Real-Time PCR is based entirely on fluorescence technology.
• The specimen is placed in an appropriate well and undergoes thermal cycling similar to conventional PCR.
• In Real-Time PCR machines, a tungsten or halogen light source excites the fluorescent marker added to the sample, causing it to emit fluorescence that increases as the DNA copy number amplifies.
• The emitted fluorescent signal is captured by a detector, converted into a digital signal, and then sent to the computer for display.
• The fluorescence becomes measurable once it rises above the threshold level, which is the lowest level of signal that the detector can reliably identify.

Fluorescence Markers used in Real Time PCR

Several markers are used in Real-Time PCR, with the most common being:

1. Taqman probe.

2. SYBR Green.

1. Taqman Probe

• A hydrolysis probe containing a reporter dye, usually fluorescein (FAM), at the 5′ end, and a quencher, commonly tetramethylrhodamine (TAMRA), at the 3′ end of the oligonucleotide.
• Under normal conditions, the probe remains coiled, keeping the fluorescent dye close to the quencher, which suppresses fluorescence.
• The oligonucleotide sequence of the probe is homologous to a region of the target gene, so it binds specifically when the target DNA is present.
• During the extension phase, Taq polymerase synthesizes the new DNA strand and its 5′ nuclease activity degrades the probe.
• This degradation separates the reporter dye from the quencher, allowing fluorescence to be emitted.
• With each cycle, more probes are cleaved, increasing the fluorescence signal, which correlates directly with the amplification of the target sequence.

2. SYBR Green

• A fluorescent dye that binds nonspecifically to the minor groove of double-stranded DNA and emits a strong fluorescent signal upon binding.
• Other dyes like Ethidium Bromide or Acridine Orange may also be used, but SYBR Green is preferred due to its higher signal intensity.
• SYBR Green is often favored over the TaqMan probe because it provides information about amplification during every cycle and also allows determination of the melting temperature (Tm) of the product, which TaqMan cannot provide.
• However, its major limitation is the lack of specificity, as it binds to any double-stranded DNA, including nonspecific products or primer dimers.

Advantages of Real-Time PCR

• Real-Time PCR offers many advantages compared to conventional PCR.
• It provides real-time insight into the reaction, helping determine which reactions have succeeded and which have failed.
• The reaction efficiency can be calculated with high precision.
• There is no need to run PCR products on an agarose gel, as melt curve analysis provides the necessary information.
• Real-Time PCR data enables truly quantitative analysis of gene expression, whereas traditional PCR was only semi-quantitative at best.
• It is faster than normal PCR.
• It reduces complexity during sample quantification.
• Unlike conventional preparative PCR, Real-Time PCR allows automatic determination of the success of multiple reactions within just a few cycles, without needing separate post-PCR analysis.
• It also minimizes the risk of false-negative results, enhancing the reliability of the assay.

Applications of Real-Time PCR

• Gene expression analysis
• Cancer research
• Drug research and development
• Disease diagnosis and management
• Viral quantification
• Food testing and safety analysis
• Detection of GMO (genetically modified organism) food
• Animal and plant breeding programs
• Gene copy number determination

References

• Principle of RT-PCR, Memorial University of Newfoundland – Biology Department
• PrimerDesign Ltd., Beginner’s Guide to Real-Time PCR (PDF resource)
• SpringerLink – Book chapter on Real-Time PCR (DOI: 10.1007/978-90-481-3132-7_3)
• SlideShare presentation by Pratyay Seth on Real-Time PCR
• Thermo Fisher Scientific – Real-Time PCR Learning Center: Essentials of Real-Time PCR
• ScienceDirect – Topic page on Real-Time Polymerase Chain Reaction