Real-time PCR: Principles and Applications

Real-time PCR also called quantitative PCR (qPCR), is a variant of standard polymerase chain reaction in which amplification and simultaneous quantitation of a target DNA is done in the same PCR machine, using commercially available fluorescence-detecting thermocyclers. Fluorescent dyes specifically label DNA of interest, and the amount of fluorescence generated is proportional to the quantity of DNA present.

Although various models of real-time PCR are available, all share common features: a standard thermocycler platform coupled with an excitation source (usually a laser or tungsten lamp), a camera for fluorescence detection, and computer and software for data processing.

Depending on the available excitation source and detection filters, a variety of fluorescent dyes may be used in qPCR. The two most commonly used real-time PCR methods are SYBR green (a dye that binds to double-stranded DNA but not to single-stranded DNA, and, when so bound, fluoresces) and TaqMan probes, respectively.

Some essential features of real-time PCR are:

• Both amplification and product detection are accomplished in one reaction vessel without ever opening. This dramatically reduces the chances of cross-contamination of samples with the amplified product.  
• Compared to conventional PCR assays, real-time PCR instruments are not only able to measure amplified product (amplicon) as it is made but are also able to quantitate the amount of product and thereby determine the number of copies of the target in the original specimen.
• The amount of time required to complete a real-time PCR assay is significantly less compared to conventional PCR-based assays because the time needed for the post-PCR detection of amplified product is eliminated by the use of fluorescent probes. Also, some systems are able to perform rapid thermal cycling based on instrument design, detecting product in as little as 20 to 30 minutes.

In general, a conventional PCR assay would require a minimum of at least 4 to 6 hours from the time that extracted nucleic acid is placed into a thermal cycler to begin amplification to subsequent product detection.

Principle

Real-time PCR is accomplished in the same manner as conventional PCR-based assays (denaturation of double-stranded DNA followed by primer annealing and extension). However, it is the detection process that discriminates real-time PCR from traditional PCR assays. In real-time PCR assays, the accumulation of amplicon is monitored as it is generated using the labeling of primers with dyes capable of fluorescence. These labels produce a change in fluorescent signal measured by the instrument following their direct interaction with or hybridization to the amplicon. This signal is related to the amount of amplified product present during each cycle and increases as the number of specific amplicons increases.

Currently, a range of fluorescent chemistries are used for amplicon detection; the more commonly used chemistries can be divided into two categories: (1) those that involve the nonspecific binding of a fluorescent dye (e.g., SYBER Green I) to double-stranded DNA and (2) fluorescent probes that bind specifically to the target of interest.

Detection Methods

A number of real-time PCR methods have been described, but two have emerged as the most popular.

PCR using SYBR

SYBR green is a dye that binds to double-stranded DNA but not to single-stranded DNA, and, when so bound, fluoresces. During PCR cycle, as more and more double-stranded product is generated to which SYBR green dye attach and fluoresces, an increasing amount of fluorescent signal is generated. The amount of fluorescence in the reaction at any particular time is directly related to the number of double-stranded DNA molecules in the reaction.

However, the downside of SYBR green is that it will bind and fluoresce all double-stranded products in the reaction, whether they are specific products, nonspecific products, primer dimers, or other amplification artefacts.

TaqMan probes were named after the popular videogame Pac-Man (Taq polymerase + PacMan = TaqMan) as the authors noticed that the exonuclease activity of the Taqpolymerase had similarities to the classic Pac-Man game.

TaqMan PCR (5’ nuclease assay)

TaqMan PCR uses dye-labelled nucleic-acid probe complementary to an internal segment of the target DNA.  This dye-labelled probe anneals to one of the template strands close to and downstream from one of the two PCR primers. The probe is labeled with two fluorescent moieties, the reporter (fluorophore) is attached to the probe’s 5’ end and the quencher is attached to its 3’ end.  

When the reporter and the quencher are connected, the quencher reduces the fluorescent signal of the reporter dye as it absorbs the energy via a fluorescence resonance energy transfer (FRET). However, during PCR, Taq polymerase, extending the primer on the probe’s target strand, displaces and degrades the annealed probe through the action of its 5’ to 3’ exonuclease function. The fluorophore is thereby released from its molecular attachment to the quencher and fluoresces.

As more PCR products are generated, the more dye-labeled probe will find target regions to join which eventually leads to release of the reporter molecule during the subsequent amplification process. The release of reporter dyes is reflected as increased intensity of the fluorescent signal which is proportional to the amount of amplicon synthesized. 

Whether using SYBR green or TaqMan probes, the relationship between signal intensity and the amount of template in a real-time PCR reaction provides a reliable means both to quantitate nucleic acids and to assay for the presence or absence of specific gene sequences.

Applications

Real-time PCR enables calculation of the starting template concentration and is a frequently used analytical tool in evaluating DNA copy number, viral load, SNP detection, and allelic discrimination. When preceded by reverse-transcription PCR, qPCR is a powerful tool to measure mRNA expression and is the gold standard for microarray gene expression data confirmation.

Advantages

Significant advantages of real-time PCR include

• its ability to measure DNA concentrations over a large range,
• its high sensitivity,
• its ability to process multiple samples simultaneously and providing immediate information.

A disadvantage is the machines are more expensive than traditional PCR machines.

References and further reading

1. Arya, M., Shergill, I. S., Williamson, M., Gommersall, L., Arya, N., & Patel, H. R. H. (2005). Basic principles of real-time quantitative PCR. Expert Reviews of Molecular Diagnostics, 5(2), 209–219.
2. John M. Butler (2012): DNA Quantitation. Advanced Topics in Forensic DNA Typing: Methodology.
3. Gibson, U. E., Heid, C. A., & Williams, P. M. (1996). A novel method for real-time quantitative RT-PCR. Genome Research, 6, 995–1001.
4. Holland, P. 1991. Detection of specific polymerase chain reaction product by utilizing the 5′->3′ exonuclease activity of Thermus aquaticus DNA polymerase.