Molecular probes and their applications in real-time quantitative PCR (qPCR)
By Andrei Laikhter
The past decades have seen a remarkable growth in the use of modified oligonuleotides as tools for the study of biological phenomena such as gene regulation, siRNA as well as for drug discovery. In particular, modified oligonucleotides have been used in a variety of applications such as antisense gene regulation and as reporter molecules in hybridization-based assays. A large arsenal of modified synthetic oligonucleotides conjugated with reporter molecules have also been employed in hybridization-based assays. The use of these methods proved to have broad applications in basic molecular biology, biochemistry research, in clinical diagnostics, and in physiology and molecular medicine. The reason for all this is the characteristic high specificity to a target nucleic acid sequence of these modified oligonucleotides. The more recently developed process of fluorescence resonance energy transfer, or FRET, has improved the performance and utility of hybridization-based assays in molecular biology and medical diagnostics. Fluorescence typically occurs from aromatic molecules, and fluorescence spectral data are generally presented as emission spectra. Furthermore, the conjugation of fluorescent substances to non-fluorescenting molecules such as oligonucleotides allows for high sensitivity detection of these molecules in vivo and in vitro. A fluorescent substance, called fluorophore, has a fluorescence lifetime and a quantum yield, which is the number of emitted photons relative to the number of absorbed photons. The characteristics of fluorophores have been outlined in the Jablonski diagram. In addition, the intensity of fluorescence can be decreased by a wide variety of processes. This decrease of fluorescence intensities is called quenching which can occur by different mechanisms. Another process of importance that occurs in the excited state is called resonance energy transfer, or RET. Whenever the emission spectrum of a fluorophore, called the donor, overlaps with the absorption spectrum of another molecule, called the acceptor, this process will occur. However, the acceptor does not need to be fluorescent. The donor and acceptor are coupled by a dipole-dipole interaction. The terms RET and FRET are commonly used to describe this process. FRET can be used to measure the distances between a donor and acceptor and Foerster distances are typically in the range of 15 to 60 Å. This distance is comparable to the diameter of many proteins and the thickness of membranes. However, the use of this methodology requires a fluorescent reporter/donor, and a chromophore or flurophore acceptor conjugated to the target molecule that may be used as a probe. The conjugate can be either another fluorophore or a quencher molecule. When one acceptor molecule is a quencher, and two chromophores are in close proximity, the energy transfer will result in relatively low levels of heat. When the distance between chromophores is considerably higher than the Forster distance (Ro), the fluorescence from a fluorophore reporter molecule will result in a much higher intensity. Therefore, assays based on FRET are typically designed in such a way that the fluorophore and quencher molecules are in close proximity. When used in the assay the chromophores become separated and fluorescence of the fluorophore can be easily registered.
Figure 1. Mode of action of FRET probes during the qPCR process
Real-time PCR is one of the most powerful hybridization-based diagnostic assay in use today (1,2). Real-time PCR allows a researcher to detect and measure the progress of PCR as reactions occur when using spectrofluorometric thermo-cyclers. This assay typically uses DNA probes, which fluoresce during the DNA amplification progresses. During the PCR process the 5’-nuclease activity of the DNA polymerase causes cleavage of the fluorogenic probe, effectively separating the reporter molecule from the quencher dye, resulting in an increase in reporter-associated fluorescence. With this system, the monitoring of the increase in fluorescence allows quantitation of the amount of PCR product amplified in a simple manner that does not require gel electrophoresis.
Figure 2. An amplification plot showing typical qPCR fluorescent profiles at various template concentrations is illustrated here. This type of detection methods is based on changes in fluorescence proportional to the increase of the target. The monitoring of the increasing fluorescence during each PCR cycle allows the user to follow the reaction in real time.
Molecular beacons are another class of DNA probes that have been successfully used for real-time PCR (3). This approach is based on fluorescence resonance energy transfer (FRET). Oligonucleotides designed in this way usually carry a fluorescein derivative (FAM) at the 5’ end as a common fluorescent donor and fluorescein or rhodamine derivatives (FAM, JOE, TAMARA, ROX, Cy3, Cy5, and others) attached to a modified thymidine within the primer sequence as a fluorescence quencher. Oligonucleotides labeled in this way make them valuable in areas where high sensitivity and spectroscopic discrimination of multiple fluorescent labels are important. Examples are automated DNA sequencing from very low amounts of template (reducing the need for cycle sequencing), DNA sequencing by hybridization in conjunction with microchip technology, in situ hybridization, and short tandem repeat detection, to name a few. These probes have a hairpin DNA structure where the fluorophore and the quencher is in close proximity to each other. When the probe hybridizes to its target complementary DNA template, both chromophores are forced away from each other and are thus generating a fluorescent signal.
Figure 3. Mode of action of molecular beacon probes during the hybridization process
A similar class of fluorogenic hairpin probes, commonly referred as “ampliflours” and “scorpions”, are also utilized as a primer in PCR reactions (4).
Figure 4. Mode of action for “ampliflour” type probes during the qPCR process
Figure 5. Mode of action for “scorpion” type probes or “scorpion probes” during the qPCR process
Yet another unique type of real-time PCR probes that was developed in collaboration with Steve Benner lab at the University of Florida is Plexor that utilizes non-natural iso-dG and iso-dC base-pairing (5,6). During the reverse polymerase reaction the dabcyl labeled iso-dGTP will be incorporated in close proximity to the fluorescently labeled iso-dC nucleo-base and subsequently will quench the fluorescence. The signal will be detected by diminishing corresponding fluorescence resulting from the incorporated forward primer.
Figure 6. Mode of action of “Plexor” type probes in the qPCR process
Several new, efficient azo-quenchers for use in DNA based probes have been reported recently. However, each of those dyes has a narrow and limited range of quenching that is predetermined by their narrow absorbance spectra. Therefore, each of those quencher dyes requires a fluorophore within a certain range of the fluorescence emission spectrum in order to have an efficient energy transfer between the two dyes. The broad absorbance spectra of our new generation of the quencher dyes such as Instant Quencher dyes (IQ4) (7) makes these probes suitable for multiplexing. The use of novel quencher dyes significantly improved sensitivity and the Ct value number of the corresponding linear probes. The Ct or threshold cycle number is found at the intersection between the amplification curve and a threshold line which is a relative measure of the concentration of target in the PCR reaction.
Figure 7. UV Spectra of standard mono-labeled decamers labeled with the leading quencher dyes
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6. Frackman, S. et al. Plexor® technology: A new chemistry for real-time PCR Promega Notes 2005, 90, 2–4.
7. Laikhter A. et al. US patent 7,956,169