Quantitative detection of specific nucleic acid sequences has become an essential tool for molecular biologists, and has proven to be especially valuable for the clinical diagnostic field and Agfood tests. Increased automation of the equipment, especially regarding the evaluation of results has been an important step towards a more general use of Real Time Amplification Kits.

Biotools, as one of the most innovating biotechnology companies, has been since the uprise of Real Time Amplifications a reference in the launch of Real Time Kits for applications in the Diagnostic, Biomedical, Agfood and Life Sciences sectors. We are continuously launching new kits to provide our customers with high quality kits.

Among our recent achievements, we have to name Biotools new LIONPROBES (registered trademark of Biotools B&M Labs, S.A.) technology (developed and patented by Biotools B&M Labs, S.A.), which is a totally new approach to Real Time DNA Amplifications, increasing sensitivity and specifity. Biotools has launched in October 2006 the first Real Time Kit based upon LIONPROBES, the BIOTUB QT Kit for quantitative detection of Mycobacterium tuberculosis. In case that you want to know more about LIONPROBES; please check our news section (home page), and you will be updated with upcoming publications and presentations about this exciting development.

Origin of Real Time Amplification

The starting point for the development of Real Time Amplification as a substitute to standard Amplification, was the demand for fast and reliable tests allowing to quantify the original amount of DNA in the sample, while offering at the same time a follow up of the reaction in real time.

Theoretically, there is a quantitative relationship between the amount of starting target sequence and the amount of amplified product at a given cycle. In practice though, it is a common experience for amplification reactions to yield different amounts of the amplified product. As a consequence, conventional (end-point) Amplification cannot be considered a quantitative assay, and the results are usually expressed in terms of positivity/negativity.

A semi quantitative evaluation is possible on the basis of the appearance of the electrophoresis band (weak/strong positivity), but this approach is highly subjective and the sensitivity is very low. To address the issue of amplified product quantitation, methods such as competitive Amplification, sequence analysis gene expression (SAGE) and high-throughput microarray have been proposed. These techniques are cumbersome, time consuming, and require multiple manipulations of the samples, thus increasing the risk of carrying over contamination.

The development of Real Time Amplification has eliminated the variability traditionally associated with Quantitative Amplification, thus allowing the routine and reliable quantitation of amplified products. This technology is based on the detection and quantitation of a fluorescent reporter. This signal increases in direct proportion to the amount of the amplified product in a reaction. By recording the amount of fluorescence emission at each cycle, it is possible to monitor the amplification reaction during the exponential phase, when the first significant increase in the amount of the amplified product correlates to the initial amount of target template.

Real Time Amplification was originally described as “Kinetic PCR” by Higuchi et al. in 1993.This “real-time” system included the intercalator ethidium bromide in each amplification reaction: as the amplification reaction progresses, double stranded DNA is synthesized and the fluorescence of ethidium bromide increases. The amount of double stranded amplified product can be monitored each cycle by fluorescence. Another double-stranded DNA binding dye widely used nowadays is SYBR Green I. The principal drawback to intercalator-based detection of amplified product accumulation is that both specific and non-specific products generate signal and certain applications require greater sequence specificity.

Real Time Amplification Systems

Real Time Amplification Systems have rapidly evolved over the last few years by fluorogenic probe-based amplified product detection. Holland et al. were the first to demonstrate that cleavage of a target probe during the Amplification by the 5' nuclease activity of DNA polymerase could be used to detect amplification of the target-specific product. In addition to the components of a typical amplification, reactions included a probe labelled with ³²P on its 5' end and blocked at its 3' end so it could not act as a primer.

During amplification, annealing of the probe to its target sequence generates a substrate that is cleaved by the 5' nuclease activity of the DNA polymerase when the enzyme extends from an upstream primer into the region of the probe. This dependence on polymerisation ensures that cleavage of the probe occurs only if the target sequence is being amplified. After the Amplification, Holland et al. measured cleavage of the probe by using thin layer chromatography to separate cleavage fragments from intact probe.

The development of dual-labelled oligonucleotide probes, by Lee et al. made it possible to eliminate post-Amplification processing for the analysis of probe degradation. The probe, so-called “TaqMan” probe, is an oligonucleotide with both a reporter fluorescent dye and a quencher dye attached, that emits a fluorescence signal only on cleavage, based on the Förster or fluorescence resonance energy transfer (FRET).

As long as the probe is intact, fluorescence energy transfer occurs in a way, in which the fluorescence emission of the reporter dye is absorbed by the quenching dye. On nuclease degradation of the probe during the Amplification, the reporter and quencher dyes are separated, and the reporter dye emission is no longer transferred to the quenching dye (no more FRET), resulting in increase of reporter fluorescence emission. This process occurs in every cycle and does not interfere with the exponential accumulation of the amplified product.

More recently, other initiatives that rely on the FRET principle, although without the need for hydrolysis by the nuclease activity of the Taq polymerase, have been developed, although their complexity and high cost excludes them at the present stage from a more important market presence:

1. Molecular beacons:

They are hairpin-shaped molecules that contain a stem-loop structure, a fluorophore and a quencher (Dabcyl). The stem sequence keeps the fluorophore and the quencher in close proximity so that any photons emitted by the fluorophore are absorbed by the quencher.

The loop sequence is complementary with the target. When the probe finds its target, the loop opens and hybridises to the target. This removes the fluorophore from the vicinity of the quencher, allowing the fluorescence to appear and be measured.

Molecular Beacons are designed to remain intact during the amplification reaction, and must rebind to target in every cycle for signal measurement. Molecular Beacons form a stem-loop structure when free in solution. Thus, the close proximity of the fluor and quench molecules prevents the probe from fluorescing. When a Molecular Beacon hybridises to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. Molecular Beacons can be used for multiplex assays by using spectrally separated fluor/quencher moieties on each probe.

2. Scorpion probes

Scorpion probes maintain a stem-loop structure in the non hybridised state, having a fluorophore attached to their 5’ end, whose emission is quenched by a moiety located at their 3’end that contains a stem with a sequence complementary to the product to be extended by the primer. One of their advantages is that they use sequence-specific priming, so that the detection of the amplified product can be obtained by using a single primer. After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended product, so that the hairpin loop is opened. This way the fluorescence is prevented from being quenched and a signal can be observed.

3. Hybridisation probes

This is a system consisting of two specifically designed, sequence-specific oligonucleotide probes, labelled with different dyes. The sequences of the probes are selected so that they can hybridise to the target sequences on the amplified DNA fragment in a head-to-tail orientation, thus bringing the two dyes into close proximity. The donor dye (fluorescein) is excited by the blue light source and emits green fluorescent light at a slightly longer wavelength. At close proximity, the energy emitted excites the acceptor dye attached to the second hybridisation probe, which then emits fluorescent light at a different wavelength. The amount of fluorescence emitted is directly proportional to the amount of target DNA generated during the Amplification reaction.

4. Intercalating dyes (SYBR Green and similar)

Despite being the most economical option for detection and quantifying real time amplified products, intercalating dyes (i.e. SYBR Green), bind to any double-stranded DNA present in the reaction, including primer-dimers and other nonspecific reaction products. This requires to take specific precautions. Therefore most of the protocols for kits based upon this technology include a melting curve analysis, intended to identify the amplified product that has been detected.