Product Survey: qPCR kits and mastermixes

Dyes or Probes?
by Harald Zähringer, Labtimes 03/2015

qPCR combines the polymerase chain reaction with fluorescent probe chemistry to directly monitor the amplicon production during each cycle. Researchers may choose from a plethora of qPCR kits and mastermixes based on different detection chemistries.

In 1992, Russel Higuchi and his colleagues introduced an enhanced PCR technique that allows detection of the amplified products without opening the reaction tubes. To this end, Higuchi simply added Ethidiumbromide (EtBr) to the PCR reaction and measured the strong increase in fluorescence, caused by the intercalation of EtBr into the amplified double-stranded DNA (dsDNA). The group plotted the fluorescence against the cycle number and obtained a graph that showed the increase of the PCR product in “Real Time”. Life scientists soon dubbed the new method “Real-Time PCR”, even though this term defies any logic, since in the real world every chemical reaction happens in real time. Real-Time PCR enables the precise quantification of the initial starting DNA and is, therefore, more appropriately, also called quantitative PCR or simply qPCR.

Since Higuchi et al.’s first qPCR experiments with EtBr, researchers have developed various other detection techniques. Most of them are commercially available in qPCR kits or mastermixes, which additionally contain a heat-stable polymerase, ­dNTPs and an appropriate buffer system. The qPCR detection chemistries used in these kits and mastermixes may be divided into two main categories: dye-based and probe-based.

Fluorescent DNA binding dyes such as EtBr, SYBRGreen, SYBRGold, EvaGreen, SYTO and Bebo to name but a few, attach to the minor groove of dsDNA, forming a dye-DNA complex that emits fluorescent light. The resulting increase in fluorescence during the extension phase of the PCR cycles is detected by a CCD camera and is further translated into an electronic signal.

DNA-intercalating fluorescent dyes, such as SYBRGreen and EvaGreen, are used in dye-based qPCR assays. Photo: Perkins Lab/University of Colorado

Cheap but unspecific

Fluorescent dyes are cheaper than fluorescent probes, however, they have the tendency to stick to every specific and non-specific PCR product, such as randomly arising primer-dimers, swimming around in the reaction pot. Hence, the specificity of the amplified products has to be checked with an additional, time-consuming, melting curve analysis. Instability and inhibition of the PCR reaction are further issues of fluorescent qPCR dyes, which should be taken into account when performing dye-based qPCR reactions.

To stay out of trouble with non-specific dyes, researchers may also quantify amplified qPCR products with target-specific fluorescent probes. qPCR probes, which are basically oligonucleotides armed with fluorescent molecules or fluorophores, are available in three different formats: primer-probes, hydrolysis-probes and hybridisation-probes.

Primer probes such as hairpins, cyclicons or angler, combine qPCR primer and probe in one molecule. Typical examples of hairpin primer-probes used in qPCR kits are scorpions, Amplifluor and LUX (light upon extension) probes. All three are composed of a single-stranded oligonucleotide with a characteristic hairpin-like secondary structure and a loop sequence complementary to the target DNA.

Hairpins and scorpions

Scorpion probes harbour a reporter fluorophore at the 5’-end of the stem sequence as well as an internal quencher and a primer, blocked by hexa-ethylene glycol, at the 3’-end. The primer binds to the target sequence and is extended during the first round of PCR. In the next denaturation step, the target-specific sequence of the loop hybridises to the newly synthesised strand.

This hybridisation event opens the stem loop and releases the quencher from the fluorophore, which in turn, leads to a strong fluorescence signal. The mode of action of Amplifluor probes is more or less identical to scorpions, except for the primer blocking, which is not required.

LUX probe reporter fluorophores are attached close to the 3’-end of the stem, to suppress the fluorescence in the free probe without a separate quencher. Integration of the LUX primer-probe into the ­­­­double-stranded target sequence exponentially enhances the emission of fluorescence light.

TaqMan probes are the most popular hydrolysis probes and presumably the most frequently employed probes in qPCR kits. They are designed to specifically bind to the target sequence and contain a donor fluorophore at the 5’-end and an acceptor fluorophore (quencher) at the 3’-end.

FRET quenching

The fluorescence of the donor is suppressed in the free probe due to Fluorescence Resonance Energy Transfer (FRET)quenching of the acceptor fluorophore. After hybridisation to the target sequence, the DNA polymerase activity of Taq extends the bound probe, while the 5’-3’-exonuclease activity of Taq removes the quencher at the 3’-end, triggering a fluorescence signal during the extension phase of the qPCR reaction.

Hydrolysis probes may also be combined with snake primers to perform a snake qPCR, in which the amplification of the target sequence and the fluorescence detection are separated into two different processes.

Molecular beacons unfold upon binding to the target DNA-sequence, which triggers the release of fluorescent light. Photo: PHRI/ Rutgers University

Snake assays

In the first step of target amplification, a forward snake primer containing a 5’-flap sequence anneals (downstream from the hydolysis primer binding site) to the sense strand of the target. The polymerase extends the primer and synthesises the antisense strand. The double-stranded amplicon is denatured at 95° C and a reverse primer binds to the antisense strand. Extension of this primer produces a sense amplicon strand with a sequence at the 3’-end complementary to the forward snake primer. The double-stranded amplicon is again separated at 95° C and the sense strand folds back into a secondary structure, similar to a hairpin with a nucleotide mismatch at the 3’-terminus (complementary to the 5’-flap).

The TaqMan probe hybridises next to the 3’-end to the sense strand of the secondary structure, creating a cleavage structure for the 5’-nuclease applied in the next stage of the snake assay. The 5’-nuclease recognises the structure, cleaves the probe and triggers a fluorescence signal.

Molecular bonfires

Hybridising probes release a fluorescence signal upon binding to the amplified target sequence. Typical examples are molecular beacons with a hairpin-like structure composed of a loop sequence complementary to the target, a stem formed by two, five to seven base pairs, a fluorescent reporter at the 5’-end and a quencher at the 3’-end. During annealing to the target sequence, the hairpin unfolds and reporter and quencher are disconnected leading to fluorescence emission.

Primer probes or probes may also be constructed using nucleic acid analogues with similar structures to naturally occurring DNA, such as Peptide, Locked and Zip nucleic acids, which opens up even more possibilities for probe-chemistry used in qPCR kits. The mode of action of these analogue probes is, however, in many cases identical to classical oligonucleotide-based qPCR probes.

First published in Labtimes 03/2015. We give no guarantee and assume no liability for article and PDF-download.

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