Fluorescence based detection is well established in Bio Sciences. In many fields it has successfully replaced radioactive isotope labeling. Fluorescent dyes have an "environmental advantage": they have a longer shelf life, are inexpensive to discard and safer to handle.
Widely used and long established as a label of antibodies in immunological detection, in fluorescence microscopy and flow cytometry, fluorescent detection found its way into molecular biology with the widespread introduction of automated DNA sequencing. The availability of instruments such as the SDS 7700 System from Applied Biosystems and the new LightCycler® Instruments from Roche Diagnostics, has increased the potential of Polymerase Chain Reaction (PCR) based on fluorescent "Real-Time" detection.
Fluorescence detection is not more sensitive than immunological or radioactive isotope detection. In fact, in most cases systems based on radioactive labeling have a ten to thousand-fold higher sensitivity.
What than are the advantages of fluorescence?
There are two major factors. One is the potential of multiple parallel measurements by using different colored dyes and the other, the potential of time resolved continuous data acquisition. Fluorescence is also detectable in a closed tube. Furthermore, the genotyping of a target sequence is possible during the amplification. Therefore, the absolute sensitivity is no longer a decisive factor for these detection methods, particularly since most applications are based on nucleic acid amplification.
Fluorescence describes the attributes of some chromophores to emit light with a longer wavelength when excited by light. The efficiency of this process is described by the quantum yield Q, a number between 0 and 1. This phenomenon is known for fluorophores spanning the spectrum from ultra violet (UV), visible (VIS) to infrared (IR) in wavelength from 300 nm to 800 nm. The absorption and emission peaks are shifted by 15 to 40 nm (Stokes-Shift).
A "good" fluorophore is characterized by strong absorption (a high extinction coefficient), high quantum yield (Q> 0.7) and a large Stokes-Shift. The light source has to harmonize with the fluorophores used. It should radiate near the absorption peak. Since the emitted fluorescence has a certain bandwidth, the different fluorophores should be separated by 15-50 nm to be detectable concomitantly. When using a monochromatic light source, the detectable dyes will have to be in close proximity due to the parallel shift of their absorption peaks. This means that, depending on the instrument, two to four fluorophores are detectable simultaneously. In the future, the differing fluorescent half-lives of diverse dyes with similar wavelengths could be used to analyze various probes simultaneously.
Fluorophores are environmentally sensitive. They are influenced by neighboring molecules, particularly by other chromophores, which may "quench" the fluorophore. This results in a reduction or complete extinction of the fluorescence emitted. On the other hand, neighboring fluorophores may transfer energy to each other, through a phenomenon known as Fluorescence-Resonance-Energy-Transfer (FRET). By means of this feature, molecular sensors can be developed, which provide information on the presence or configuration of molecules. This allows continuous observation and analysis of a reaction avoiding tedious washing-off or separation of reaction components, as in all other conventional assays that rely on binding. Examples for this type of "conventional" method are: Northern-and Southern-Blot, reverse blot techniques, PCR-ELISA on micro titer plates or membranes, cDNA arrays or DNA-chips and their detection with radioactive labeled markers or haptens and enzyme-conjugated antibodies, e.g. Digoxigenin-Systems.
The continuous detection of the nucleic acid amplification process allows totally new potentials in the quantification of nucleic acids. The quantity of target nucleic acids is relevant in several topic areas. For example, in virology where the determinations of a virus load will help to establish the success of an antiviral therapy. In cases of leukemia it helps to document the therapeutic success and monitor Minimal Residual Disease. It allows the supervision of the reproduction of donor cells in post bone marrow transplantation patients. It helps to establish the composition of foodstuffs, e.g. the quantity of genetically modified constituents.
In the past, quantitative PCR analysis was limited to an estimation of the amplicon quantity in a gel electrophoresis or by titration in the presence of a competitor PCR. The continuous fluorescence detection allows for a much simpler determination of the template quantity. The number of PCR cycles necessary to reach a given product quantity is over a wide range proportional to the starting quantity, having generally a linear range of four to seven logs. The data is collected and analyzed during the PCR reaction. The results are presented at the end of the amplification, omitting additional pipetting steps, eliminating the risk of PCR product contamination and avoiding gel electrophoresis.
Real-Time PCR Formats
There are different possibilities of creating reaction-dependent fluorescence signals. The simplest technique is the use of a dye that when bound to double stranded DNA will fluoresce (Ethidium bromide, SYBRGreen). However, the signal is not truly specific. These dyes will also detect primer-dimers and false amplicons.
There are a variety of probe types, which, by making use of FRET or quenching, will create a fluorescent signal in the presence of their target.
TaqMan Probes, Hydrolysis Probes, 5’-Nuclease Process
TaqMan probes are single-stranded oligonucleotides that can bind to the amplicon and are modified to contain a fluorophore and quencher 3 to 30 bases apart. During the amplification, the probe will be hydrolyzed by a double-strand specific nuclease activity associated with the Taq Polymerase resulting in a dissociation of reporter from quencher – the fluorescence increases.
TaqMan probes are relatively sensitive to single base variations (mismatch). This could be extremely important when amplifying virological samples, where such a genetic variability could be present that a successful amplification may fail to result in a positive signal. Unfortunately, this sensitivity may render TaqMan probes inappropriate for genotyping, since a ‘non-signal’ will have to be attributed to a genotype.
There are various possibilities to allow adjacent hybridization of oligonucleotides. Probably the most popular and successful method is the binding of two single labeled probes in a head to toe manner, also known as "kissing probes" or HybProbe. The FRET induced fluorescence of the acceptor dye is detected and measured. An advantage over the hydrolysis probes is their modular assembly and for quantitative PCR, their relative robustness towards single base variations; but, probably the most outstanding feature is their excellent suitability for genotyping. A disadvantage is the need for a larger sequence area necessary to accommodate two adjacent probes.
A variation of the preceding format is the replacement of one of the probes by a labeled amplification primer. A labeled probe that binds to the strand containing the extended primer provides the FRET reaction necessary for the detection.
Furthermore, self-complementary oligonucleotides, one labeled with a fluorophore the other with a quencher have been used to monitor a PCR reaction. The increase of target concentration causes a primer binding equilibrium that distances the quencher from the reporter.
A very interesting variation of hybridization probes is Molecular Beacons, developed by Fred R. Kramer. The ends of the probes are self-complementary and labeled with a fluorophore/quencher pair. In absence of a complementary sequence, these molecules fold into a stem-loop structure and the fluorescence is extinguished by the quencher. When bound to a target the increased distance between quencher and dye results in an increase of detectable fluorescence. Molecular Beacons are sensitive to mismatches.
Sunrise primers are similar to Molecular Beacons. They are self-complementary and dissociate through the synthesis of the complementary strand. It’s in their nature to produce fluorescence signals due to unspecific products and primer-dimers.
The analysis of polymorphisms for genotyping is eminent.
Examples are paternity affiliation, species determination, the presence of genetic risk-factors and detection of disease responsible alleles. SNP studies with TaqMan probes or Molecular Beacons can only be carried out with different probes targeting all known variations. When labeled with different dyes the analysis may be carried out in the same reaction assay. On the other hand, HybProbes have the decisive advantage of allowing the detection of mutations with only one specific probe, with a melting curve analysis thru the lowering of the melting temperature induced by the mutation. In a homozygous context a single high or lower temperature transition is detected, in a heterozygous background two temperature transitions will be displayed.
The new Real-Time PCR technologies allow a much faster investigation of samples; the qualitative detection of nucleic acids, as well as their quantification and genotyping
The time for an analysis shrinks to 20 to 30 minutes with the LightCycler®. The reduction of steps from sample preparation to result interpretation will render PCR analysis less expensive, consequently increasing the use of PCR in diagnostics.
Hapten modified Probes
Hapten modified probes are commonly used for specific detection through hybridization to nucleic acids on immobilized membranes, in vitro and in situ. The detection technique is based on the interaction of molecules with high binding affinity to the haptens (e.g. Streptavidin, Antibodies) which in turn are conjugated to Enzymes (Peroxidases, Phosphatases). In a final incubation step the added enzyme substrate is catalyzed into a detectable/measurable product.
In most cases, they represent a good substitution for isotope labeled probes.
Fluorescent labeled Probes.
TIB offers a wide variety of fluorescent labels spanning the spectrum from near UV to infrared. The applications of fluorescent labeled probes range from automated sequencing, over micro-satellite analysis, to in situ hybridization. The price includes the DNA sequence up to 30 bases, all necessary modifications, and purification by HPR3 or FPLC. Products are delivered desalted and lyophilized accompanied by a detailed data sheet.
TaqMan is a trademark of Hoffmann-La Roche, SYBR Green is a trademark of Molecular Probes, LightCycler® is trademark of Hoffmann-La Roche. PCR and the 5'-Nuclease-Procedure are patented by Hoffmann La-Roche. Molecular Beacons is patented by PHRI. The Sunrise Principle is patented by Onkor. TIB MOLBIOL is a licensed producer of HybProbes for the LightCycler® and LightTyper® Systems, the production of Molecular Probes and a large number of other fluorophore-coupled probes and primers.