Optimized qPCRs with AAT Bioquest

Written by Emily Locke

These topics await you:

1) The Successful Recipe behind (q)PCR

2) Dye-based vs. Probe-based qPCR

3) Selection of Fluorescent Reporter Dyes

4) Selection of Quencher Dyes

5) Recommended FRET Pairs for developing FRET Oligonucleotides

6) ROX - the Important Player in the Background

7) AATs Mastermixes for more Convenient qPCR Assays

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On a moonlit Friday night in April 1983, Kary B. Mullis got into his car and set off on a drive through the mountains of California. At the time, he was certainly unaware that this moment would eventually revolutionize the entire field of molecular biology. It was during this nocturnal drive that Mullis came up with the idea for the polymerase chain reaction (PCR) - probably the most widely used method in molecular biology today [1]. PCR has become an absolute standard tool for researchers, but since the COVID pandemic at the latest, even non-scientists have become familiar with the method, which allows DNA to be amplified quickly and cheaply. At the time, Mullis received only a $10,000 bonus from his employer, while the rights to the PCR were sold shortly after to Roche for $300 million. He finally received the well-deserved recognition for his groundbreaking invention in 1993 with the Nobel Prize in Chemistry [2].

In the meantime, many different variants of PCR have become established for a wide variety of applications. One of the most important and most widely used methods besides conventional PCR is real-time quantitative PCR (qPCR). This further development of standard PCR enables not only the amplification of DNA but also the quantification of the amplified genetic material and is used, for example, in gene expression studies, in the discovery of biomarkers or the measurement of RNA interference [3]. To ensure that you are well equipped for your future qPCR experiments, our supplier AAT Bioquest from California (pure coincidence) provides you with high-quality bioanalytical reagents. The company is a specialist in absorption, fluorescence and luminescence technologies. Therefore, wherever fluorescence and luminescence are used, you can rely on products from AAT - including your qPCR experiments. 

The Successful Recipe behind (q)PCR

But first, let's take a big step back - how exactly does PCR work again? In principle, the recipe for the successful procedure is quite simple and consists of only three steps: Denaturation, primer hybridization and elongation (Fig. 1). In the first step, the double-stranded DNA template (dsDNA) is heated to 95 °C, which breaks the hydrogen bonds between the complementary base pairs and separates the DNA strands (denaturation, Fig. 1.1). In the second step, the primers can anneal to the now single-stranded DNA at lowered temperature (annealing, Fig. 1.2) with the exact annealing temperature depending on the length and sequence of the primers. The primers bind to specific regions within the DNA template so that only a specific sequence (sequence of interest) is amplified. This amplification is achieved by the DNA polymerase, which, starting from the 3' end of the attached primer, synthesizes the new strand complementary to the single-stranded DNA template (extension, Fig. 1.3). Such a cycle is repeated approximately 20 to 50 times during PCR, resulting in exponential amplification of the desired target sequence [4].

Figure 1: Process of a polymerase chain reaction. DNTPs, specific primers and a DNA polymerase are added to the DNA template containing the sequence to be amplified. In the first step, the double-stranded DNA is heated and thus separated into single strands (1). This is followed by primer annealing (2) and extension by the polymerase (3). The cycles are repeated 20 to 50 times, resulting in an exponential amplification of the target sequence [5].

The principle behind the amplification of DNA in a qPCR is analogous to that of a conventional PCR. The major difference between the two variants is that qPCR allows quantification of the amplified DNA using fluorescence measurements acquired in real time during a PCR cycle. Fluorescence increases proportionally with the amount of PCR products and is monitored by a thermal cycler throughout the PCR run [3]. By plotting the intensity of the fluorescence signal against the cycle number, qPCR instruments can generate so-called amplification plots, which show the accumulation of amplicons throughout the PCR run (Fig. 2). The so-called Ct value (cycle threshold) or Cp value (crossing point) describes the cycle at which fluorescence first rises significantly above background fluorescence. Often, a household gene is used as an internal control to perform a relative quantification according to the ΔΔCt method. To do this, the Ct values of the target gene and reference are subtracted from each other and then the difference between the ΔCt values of each group is formed. By substituting into the formula 2^-ΔΔCt, the relative gene expression to the reference is obtained [6].

Figure 2: Typical amplification plot of a qPCR. The graph shows the accumulation of amplicons during a PCR run. The target sequence in this example was GAPDH, using various amounts of cDNA (ranging from 100 ng to 0.00001 ng). AAT's Helixyte™ Green *20X Aqueous PCR Solution* was used to detect the PCR products [3].

Dye-based vs. Probe-based qPCR - Two Methods in Comparison

And where does the fluorescence come from? In general, there are two different strategies in qPCRs that allow the determination of DNA quantities: dye-based (Fig. 3) and probe-based (Fig. 4) qPCRs [3]. The dye-based method uses a fluorophore that intercalates into the DNA by binding to the minor groove of the dsDNA (Fig. 3.3). The dye shows a weak background fluorescence, the intensity of which increases significantly by binding to the dsDNA. Amplification of the target sequence creates more binding sites for the dye, which is why the increase in fluorescence directly correlates with the amount of dsDNA present [7].

Figure 3: Principle of dye-based qPCR. Denaturation (1) and primer annealing (2) are followed by extension of the target sequence by DNA polymerase (3). The intercalating dye binds to the small groove of the newly formed dsDNA. An increase in the amount of DNA during PCR therefore leads to an increase in the fluorescence intensity measured at each cycle [3].

For the dye-based method, only two sequence-specific primers are required, making this variant the fastest and most cost-effective option for qPCR. However, the intercalation of the dye is not limited to the target sequence, which means that unwanted products and primer dimers can also be detected, which in turn can result in inaccurate quantification. Therefore, to ensure that only the desired target has been amplified, one usually generates a melting curve after each experiment, which can be used to determine the accuracy of the reaction. Furthermore, with the dye-based method, only one target sequence can be quantified at a time; multiplexing measurements are not possible [7].  

The inaccuracy of the dye and the limitation to one target gene can be overcome with probe-based qPCR (Fig. 4). Here, the fluorescence of a fluorophore-labeled, sequence-specific probe is measured in real time. Probe designs can vary widely, with so-called hydrolysis probes being the most common. These are short oligonucleotides whose sequence is complementary to the target sequence and are labeled with a 5' reporter fluorophore as well as a 3' quencher. As long as the oligo probe is intact, it emits only a small amount of fluorescence because Förster resonance energy transfer (FRET) between the reporter and quencher prevents emission of the fluorophore (Fig. 4.2).

Figure 4: Principle of probe-based qPCR. Denaturation (1) and primer annealing (2) are followed by extension of the target sequence by DNA polymerase (3). During synthesis of the opposite strand, the probe is degraded at the 5' end, causing the quencher and fluorophore to move away from each other. The resulting increasing reporter fluorescence can be measured at the end of elongation in each cycle [3].

Since the DNA polymerase used in PCR also has an endonuclease domain, the probe is hydrolyzed by this domain when the target sequence is amplified, and the fluorescent reporter is cleaved from the oligonucleotide [8]. This results in spatial separation of the reporter fluorophore from the quencher, leading to an amplification-dependent increase in fluorescence (Fig. 4.3). The probe-based strategy is often the preferred technology for diagnostic applications because it is typically more specific than dye-based qPCRs. In addition, multiplexing measurements are possible by using a different fluorophore for each target. However, this method is much more expensive and laborious compared to the dye-based technology [7].

*other available variants: acid, alkyne, amine, azide, maleimide

*other available variants: acid, alkyne, amine, azide, maleimide

ROX - the Important Player in the Background

In addition to the reagents already mentioned (DNA template, sequence-specific primers, DNA polymerase, intercalating dye or probe), there is another essential component that should not be missing in your qPCR master mix: the passive reference dye ROX or carboxy-X-rhodamine [9]. This fluorophore is used to normalize the intensity of the fluorescent signal from the reporter dye. This makes it possible to compensate for non-PCR-based variations in the fluorescence signal, such as pipetting errors, air bubbles, condensation, or drift of instrument parameters [10]. Since ROX does not interfere with the qPCR reaction and has an easily recognizable red emission spectrum, it provides an excellent baseline in multiplex qPCR assays. The reference dye thus contributes significantly to the accuracy and reproducibility of qPCRs and is therefore used in many laboratories [3].

Despite its wide range of applications, ROX has one or the other drawback: the reference dye shows only low stability and also needs to be stored cold to maintain its fluorescence. Therefore, our partner AAT Bioquest has developed a new reference dye: 6-ROXtra™ shows increased stability as well as improved water solubility compared to conventional ROX and still has similar spectral properties [3]. 

AAT's Mastermixes for more Convenient qPCR Assays

All researchers who have ever performed qPCR know of the effort involved. It is especially high if you want to test many different samples. But don't worry - AAT has a suitable solution for this as well, providing you with convenient, premixed cocktails that contain all the necessary components for a qPCR (except template, primer and probes, if applicable). The TAQuest™ Master Mixes simplify set-up without compromising sensitivity, specificity, or PCR efficiency. The mixes contain a TAQuest™ Hot Start Taq DNA polymerase, which allows the reaction to be set up at room temperature, as well as dNTPs, MgCl₂ and stabilizers in an optimized reaction buffer. Mixes are also available with Helixyte™ Green, a DNA-intercalating dye that enables detection of PCR products during amplification (see Fig. 2). In addition, you can choose mixes with a ROX reference dye for increased data precision as well as for troubleshooting support [3].

Curious for more products from our fluorescence specialist? Here you can find the complete portfolio of AAT Bioquest. And while you are already browsing: Take advantage of our 10% discount on all labeling kits from AAT!

Sources

[1] Mullis, K. B. The Unusual Origin of the Polymerase Chain Reaction. Scientific American. 262(4), 56-65 (1990).

[2] https://www.haematopathologie-hamburg.de/methoden/pcr-qpcr/, 03.06.2023.

[3] https://www.aatbio.com/catalog/real-time-pcr-qpcr, 03.06.2023.

[4] https://de.wikipedia.org/wiki/Polymerase-Kettenreaktion, 03.06.2023.

[5] https://commons.wikimedia.org/wiki/File:Polymerase_chain_reaction-en.svg, 03.06.2023.

[6] https://de.wikipedia.org/wiki/Real_Time_Quantitative_PCR, 03.06.2023.

[7] https://www.neb-online.de/pcr-dna-amplifikation/qpcr-real-time-pcr-und-rt-qpcr/, 03.06.2023.

[8] https://portlandpress.com/biochemist/article/42/3/48/225280/A-beginner-s-guide-to-RT-PCR-qPCR-and-RT-qPCR, 03.06.2023.

[9] https://www.genaxxon.com/shop-all-products/chemikalien/fluoreszenzmarker/3798/5/6-rox-isomerengemisch-100-mg-s5411.0100, 03.06.2023.

[10] https://de.lumiprobe.com/p/rox-reference-dye, 03.06.2023.

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