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Real-Time PCR or quantitative PCR (qPCR) is a PCR-based technique that is able to simultaneously amplify and detect changes in the amplicon concentration. Real-time PCR collects data during PCR amplification by utilizing fluorescence signals emitted by either special probes or DNA binding dyes (1)(2)(3)(4).
Real-time PCR was first reported by Kary Mullis in 1985 when a radioactive isotope 32P was used to label complimentary probes that hybridized to PCR products (5). Later methods included the addition of Ethidium Bromide (a DNA binding dye that binds to the minor groove of the double helix structure) to PCR which later provided the basis for the development of the SYBR Green method (4). In Real-time PCR methods, however, DNA binding dyes would bind to non-specific amplicons such as primer dimers and/or sample cross contamination. Therefore, a new method was invented that utilized the 5’ to 3’ exonuclease activity of Taq DNA Polymerase coupled with special probes that are labeled at both 5’ and 3’ ends. This method is known as the TaqMan probe method and increases the likelihood that the fluorescence observed is only from the target amplicon (6)(7). Even though the starting material for real-time PCR is DNA, coupling a reverse transcription step before qPCR allows quantification of RNA samples as well (7) (8). Read more about RT-qPCR on our Reverse Transcription Knowledge Base.
The ability to detect fluorescence signals is crucial to the proper functioning of qPCR. Proper instrumentation is required for both energy input (to excite fluorescent dyes) as well as detection of wavelengths emitted by these dyes. Three main ways an instrument provides excitation energy are: lamps (ABI Prism 7000, Bio-Rad iCycler iQ), LEDs (Roche LightCycler), and lasers (ABI Prism 7900HT). Data is collected via photodetectors that only allows passage of desired wavelengths (the peak of the fluorophore's emission spectrum) in a single reaction tube (Figure 1). The last portion of the instrument consists of a regular thermal cycler for PCR amplification. Appropriate computer software is required for the proper collection and analysis of data (2).
As data is being collected during thermal cycling, the analysis software produces an amplification plot (Figure 2). At earlier amplification cycles, the fluorescence emitted by the fluorescent dyes is still well below the detection limit of the instrument; therefore, a baseline is shown. Amplification plots are essentially log graphs with ∆Rn plotted against the cycle number. ∆Rn is calculated using the equation: ∆Rn= Rnf-Rnb where Rnf is the fluorescence emission at any given point and Rnb is the baseline fluorescence emission. The CT value is the cycle number at which the fluorescence signal (∆Rn) of a given sample crosses the threshold value given by the software or decided by the user. The CT value is inversely proportional to the starting concentration of DNA material. DNA amplicons are doubled after every CT value until the reaction reaches a plateau. Therefore, CT values are used for absolute and relative quantification of DNA and RNA (1)(2)(7).
The Theory
TaqMan Probes are designed to bind to target sequences on the amplicon. It contains a fluorescent reporter dye attached to the 5’ end and a quencher dye that is attached to the 3’ end of the probe. The proximity of the two dyes inhibits the reporter from emitting fluorescence. The Taq DNA polymerase used in the TaqMan method has 5’ to 3’ exonuclease activity which allows cleavage of the 5’ terminal nucleotide. As the Taq DNA polymerase amplifies the DNA strand extended from the primer, it encounters the probe that is hybridized and displaces the 5’ reporter dye. The Taq DNA polymerase then cleaves the reporter and relieves the reporter from being quenched. Fluorescence emitted by the reporter will be detected by the thermal cycler and recorded (9) (10) (12) (Figure 3).
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Fluorescence of TaqMan Probes
Fluorescent dyes or fluorophores are excited by photons (i.e. UV rays). When the photon is absorbed by the fluorophore’s electrons, the photon brings the electrons from a ground state to an excited state. In order to return to ground state, the energy is emitted by the fluorophore through a lower energy wavelength that is different than the excitation wavelength (Figure 4). The difference in absorbance and emission wavelengths of the fluorophore is due to the partial energy loss before emission (13).
Quenching and fluorescence of the probes are mainly explained by two mechanisms: Fluorescence resonance energy transfer (FRET) or non-FRET static quenching. In FRET quenching, when the donor fluorophore is excited by a photon, it transfers the energy to the acceptor fluorophore through dipole-dipole interactions instead of emitting the excitation energy directly. This energy transfer quenches the donor fluorophore and excites the acceptor fluorophore as a result. In qPCR, if both the donor fluorophore (reporter) and acceptor fluorophore (quencher) are able to emit fluorescence, the data will be more difficult to interpret. Therefore, quencher dyes in TaqMan probes are usually "dark" quenchers that do not emit their own fluorescence (31)(14).
In non-FRET static quenching, the reporter and quencher are in close proximity to each other. The physical interaction between the two chemicals brings the reporter down to a ground state. And this in turn, quenches the reporter fluorescence (Figure 5) (10)(12).
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Probe Designs
TaqMan probes are hydrolysis probes that utilize the 5’ to 3’ exonuclease activity of DNA polymerase. They are dually labeled with a fluorescent dye (fluorophore) on the 5’ end and a quencher dye on the 3’ end. Several different types of reporter dyes can be used in TaqMan probes, including FAM, TET, or JOE, and different types of quenchers can be used as well, including TAMRA, BHQ and MGB (13)(14). Choosing different reporters will depend on the instrumentation used for the experiment. Also, if different reporter dyes are used in multiplex experiments to detect multiple oligonucleotide sequences, the emission maxima (the peak of emission spectrum) of reporter dyes must have a difference of at least 15 nm. When choosing the quencher dye that pairs with the reporter, one must consider both FRET and static quenching. Since FRET works by transferring energy from the reporter to the quencher, it is essential for the emission spectrum of the reporter to overlap with the absorbance spectrum of the quencher. In static quenching, the structure of the dyes is crucial when pairing the quencher and the reporter (13)(14)(15).
The sequences of TaqMan probes should be designed with the following considerations:
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DNA polymerase
The DNA polymerase used in the TaqMan method possesses 5’ to 3’ exonuclease activity that cleaves double-strand DNA (dsDNA) at the 5’ terminal nucleotides and releases the oligonucleotides (16)(9). The DNA polymerase extends the primer sequence and as it synthesizes the new complementary strand, it will encounter the TaqMan Probe (9). Taq DNA polymerase prefers to cleave displaced single-stranded DNA (ssDNA), therefore, TaqMan Probe will be degraded by the polymerase thereby releasing the fluorophore into the reaction mixture and relieving the quenching effect. This allows fluorescence to be detected by the thermal cycler (12).
The Theory
The SYBR Green method depends on the intercalating dye, SYBR Green I. When this dye binds to double-strand DNA (dsDNA), it emits a fluorescent signal which can be detected by the thermal cycler. Since SYBR Green I is a non-specific dsDNA binding dye, it will bind to all dsDNA fragments present in the reaction (17)(18)(1).
When unbound to dsDNA, the SYBR Green dye does not emit very much fluorescence, however when the DNA polymerase synthesizes the complementary strand to the template, SYBR Green I incorporates itself into the dsDNA and emits a strong fluorescent signal. At the start of the next PCR cycle, the fluorescent signal is decreased when the dsDNA is denatured. Therefore, the fluorescent signal of the sample is collected at the end of the elongation step of each PCR cycle to determine the relative change in amplified products (1) (Figure 6).
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Melt Curve Analysis
Due to the chemistry of SYBR Green I Dye, users are unable to distinguish between desired products and non-specific products during the PCR cycling stage. By incorporating a melt curve analysis after PCR, users can distinguish the amplified products through plotting fluorescence as a function of temperature.
When DNA is heated, it weakens and eventually breaks the hydrogen bond that holds the two ssDNA molecules together; this process is called DNA melting or denaturation. Melting temperature or Tm is defined as the temperature at which the DNA molecule is in mid-transition between the fully coiled and fully denatured states (27)(28). Different DNA products have different melting temperatures. When the DNA template denatures, the SYBR Green I Dye incorporated in the template is released into solution and the fluorescence decreases (1) (18) (20). Unlike gel electrophoresis where one can only differentiate different products based on their product sizes, melt curve analysis can distinguish between amplified products with similar lengths. This is possible because different PCR products will have different GC/AT ratios. Even when GC/AT content is similar in both amplicons, the distribution of GC and AT will also alter the melting temperature.
DNA polymerase
SYBR Green I fluorescence is dependent on the binding of the dye to the dsDNA, therefore less restrictions apply to DNA polymerases that can be used with this method. Though 5’ to 3’ exonuclease activity is not needed in this case, Taq DNA polymerase can still be used for SYBR Green I based qPCR. Since extensive thermal cycling is used in qPCR, DNA polymerases used in this method must be thermostable (1). Another consideration when choosing DNA polymerases is that certain chemicals from forensic, food or clinical samples contain PCR inhibitors. In such cases, alternatives such as Tfl, Pwo, Tth, and Pfu DNA polymerases that are resistant to these inhibitors are available (1)(2)(22)(23)(24).
One of the major differences between SYBR Green and TaqMan methods is the use of special hydrolysis probes in the reaction (18). While SYBR Green fluorescent dye binds to all DNA molecules in the reaction, the special probes from TaqMan method only anneal to target amplicons. The TaqMan Method decreases the chance that non-specific and contamination products in PCR are interpreted as the target amplicon in the reaction, therefore dramatically increasing the specificity of the reaction (7)(1). Also, by incorporating different fluorophores into the hydrolysis probes, different reporter signals may be detected by the thermal cycler, thus allowing multiplex reactions (the ability to monitor different amplicons in a single reaction simultaneously) (1). A downside to TaqMan based qPCR is that since 5’ to 3’ exonuclease activity is crucial in the proper functioning of this assay, there are little to no alternatives to compensate for signal loss when samples contain DNA polymerase inhibitors (2).
The SYBR Green method is much more readily available, as this method is less expensive (1). However, one setback is that the fluorescent dye may bind to both target amplicons and other non-specific sequences (1)(2). Hence, an amplification plot is not enough to distinguish fluorescence signals from these different sources. This problem can be solved by using a melt curve analysis which is sensitive enough to detect different amplicons with Tm’s just 2 ℃ apart (19). This analysis, in some cases, also gives SYBR Green method the capability to perform multiplex reactions (1). Another benefit for using the SYBR Green method is the variety of DNA polymerases that can be used in the reaction. If one DNA polymerase is inhibited, many alternatives can be utilized to bypass the inhibition (21).
Over the years, qPCR has been utilized for many applications, including research and diagnostic purposes. Since qPCR is able to quickly and accurately amplify target DNA templates, it is mostly used for quantitative analysis of gene expression in the research setting. Both absolute and relative gene expression can be determined by the Ct values. Other applications in the research setting include, validating DNA microarray results, and the quantification of viral, bacterial, or fungal loads. qPCR provides researchers a cheaper and quicker alternative to sequencing when genotyping work is required in their research. This is accomplished by performing melting point analysis on the PCR products with the help of special hybridization probes. When mutations occur, the fluorescence will decrease at a lower temperature than wildtype (2).
qPCR is now being used more and more in diagnostics. As mentioned above, qPCR is able to count viral load in a given sample. Clinical diagnostic laboratories can use qPCR to evaluate disease progress in patients and evaluate the competence of anti-viral therapies (25). qPCR is also able to detect a wide range of bacterial infections in patients (including, Mycobacterium tuberculosis, Legionella pneumophila, Listeria monocytogenes, and Neisserua gonorhoeae) as well as antibiotic resistant in many strains (Staphylococcus aureus, Staphylococcus epidermidis, Helicobacter pylori, Enterococcus faecalis, and Enterococcus faecium) (2).
Finally, forensic testing is another popular application for qPCR. The specificity, sensitivity, and rapidness of qPCR enables effective detecion of small amounts of DNA sample and determines the extent of DNA degradation in a time sensitive matter (2)(26).
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